The Queen thanked all those who fought for their "heroism, courage and sacrifice of those who lost their lives".
"Seventy-five years ago, hundreds of thousands of young soldiers, sailors and airmen left these shores in the cause of freedom," the Queen said.
"In a broadcast to the nation at that time, my Father, King George VI, said: 'What is demanded from us all is something more than courage and endurance; we need a revival of spirit, a new unconquerable resolve'.
"That is exactly what those brave men brought to the battle, as the fate of the world depended on their success."
Considered a turning point in the Second World War, Operation Overlord saw thousands killed and injured after it launched on 6 June 1944.
World leaders representing the Allied nations who took part in the D-Day landings also attended, including French President Emmanuel Macron and Prime Minister of Canada Justin Trudeau.
Other guests included Australia's Prime Minister Scott Morrison, Prime Minister Charles Michel from Belgium, the Czech Republic's Prime Minister Andrej Babis and President Prokopis Pavlopoulos from Greece. Chancellor Angela Merkel represented Germany.
The PM of Luxembourg Xavier Bettel also attended, as did his counterparts from the Netherlands Mark Rutte, Norway's Erna Solberg, Poland's Mateusz Morawiecki and Slovakia's deputy prime minister Richard Rasi.
They all met the Queen before the event began and then posed for a group photograph with the monarch and Prince of Wales.
Mrs May is making her final official appearances as the British Prime Minister during the D-Day commemorations which continue on Thursday across Normandy.
At the event, the Prime Minister called for continued Western unity in tackling "new and evolving security threats" as she commemorated the 75th anniversary of D-Day.
The event is the first time the UK has hosted this many world leaders outside a formal summit since the 2012 Olympics.
Representatives from every country that fought alongside the UK in Operation Overlord - the Battle of Normandy - attended commemorations alongside the Queen, the Prince of Wales, members of the armed forces and more than 300 veterans who are all over 90 years old.
The Chief of the Defence Staff, General Sir Nick Carter, said the event was "uplifting".
"There were many things that brought a slight tear to your eye," he said.
"I always find that particularly moving piece of oratory by Winston Churchill about fighting on the beaches a very very moving experience."
Mrs May was joined by US President Donald Trump, French President Emmanuel Macron and German Chancellor Angela Merkel, as well as prime ministers from Canada, Australia, New Zealand, Belgium, the Czech Republic, Greece, Luxembourg, the Netherlands, Norway, Poland, Slovakia and Denmark.
A guard of honour formed of military personnel from the Royal Navy, Army and RAF marched through the spectator aisles and on to the main stage.
Musicians from the Band of the Royal Marines played a fanfare when the Queen arrived in the royal box, with the Tri-Service Orchestra performing the national anthem.
The event on Southsea Common in the Hampshire port city also included an hour-long production telling the story of the invasion with testimony from veterans, theatrical performances and live music, as well as a flypast of the Red Arrows and Spitfires.
As part of the performance, Mrs May is expected to a letter written by Captain Norman Skinner of the Royal Army Service Corps to his wife Gladys on 3 June 1944.
The letter was in his pocket when he landed on Sword Beach on 6 June 1944.
He was killed the next day, leaving behind his wife and two young daughters.
From the deck of the aircraft carrier HMS Queen Elizabeth, Mrs May and Defence Secretary Penny Mordaunt will wave off some 300 veterans as they retrace the journey they made across the Channel 75 years ago, followed by a flotilla of Royal Navy vessels.
More than 4,000 personnel will be involved in D-Day events in the UK and France, in what is set to be one of the biggest mobilisations of the UK armed forces in recent history.
On Thursday, Mrs May will commemorate the anniversary in Normandy at the inauguration of a memorial to British servicemen at Ver-sur-Mer, overlooking Gold Beach, as well as attending services of remembrance at the cathedral and cemetery in Bayeux.
Speaking at the event, the Defence Secretary remarked on the importance of working as a community in order to face threats.
Penny Mordaunt referenced to the specific example of the city of Portsmouth on D-Day and the efforts and sacrifices made by troops and civilians alike.
"If we come together, we work together. If we have faith in each other, we can do incredible things."
Discussing the inspiration the UK can take from the military tactics used on D-Day, the Defence Secretary said it was important to keep in mind how "people were constantly innovating, changing what they did," in order to understand the threat in a better way.
"We have different threats facing us, not just from states, but from terrorists, organisations and states using proxies to carry out attacks on us, asymmetric warfare," said Ms Mordaunt.
"What we have to do... is always thinking about tomorrow and what threats we might face around the corner."