Members of England's lesbian, gay, bisexual and transgender (LGBT) communities turned out in force in London's Soho district, with other towns and cities also staging memorials.
In London, the Pride flag was flown at half mast from City Hall in memory of the 49 people killed by a suspected terrorist who opened fire inside a gay nightclub on Sunday.
Mayor Sadiq Khan said the capital stood "shoulder to shoulder" with Orlando, ahead of vigils around the country.
Memorials have also been held in towns and cities including Manchester, Leicester, Brighton and Nottingham, with others due to stage events later.
A huge crowed gathered outside the Admiral Duncan pub in Old Compton Street, Soho, where three people died and many more were injured in a bomb attack in 1999.
People packed into the street and surrounding roads to show their support.
Rainbow flags, posters and banners emblazoned with slogans such as  "Love wins" and "When gay and trans lives are under attack, fight back" were dotted around the crowd.
At 19:00 BST exactly, a klaxon signalled the start of the vigil and those gathered immediately descended into silence.
Some hugged those close to them while others bowed their heads in memory of those who died in Orlando, and also in other attacks on gay communities around the world.
It was billed as being a two-minute silence, but it went on for far longer. Afterwards, people cheered and clapped before chanting: "We're here, we're queer, we will not live in fear."
An emotional rendition of Simon and Garfunkel's hit Bridge Over Troubled Water was then sung by London Gay Men's Chorus.
Jennifer Dean, from Bromley, said she was moved by the vigil outside the Admiral Duncan pub, where three people were killed in a nail-bomb attack in 1999.
"I just had to come here. I had to attend here and be counted and to show support and let Orlando know that they're not alone.
"I've experienced hatred and it was an attack on friends.
"It was very emotional for me. I was surprised by how many people are here. It shows respect and joy and defiance and that we're not going to be pushed around."
Martin Wilson, from Greenwich, said he "wanted to show the world that peace is important".
"Gay people have been victimised around the world and I want to show those countries that those people can be happy and free.
"The vigil was very well done, it was a fantastic event. It was important that it was held here outside the Admiral Duncan pub - they couldn't have picked a better location."
Nicholas Hall, an American who has been studying in London for last nine months, used to live in Miami and knew people who had witnessed the shooting.
"I had many acquaintances in the Pulse nightclub, four of five of my friends and a lot of people they knew," he said.
"Seeing the victims ages, in their 20s, 30s - my age - brought it much closer to home.
"I'm definitely nervous, I wouldn't say there's going to be attacks but it's made me more security aware - It hasn't put me off showing affection in public.
"The reality is hitting home to be more vigilant in London."
Ash Cox travelled from Colchester, Essex, armed with a list of the names of some of those killed in the Orlando massacre.
"When I read the news, it was very shocking and emotional. It was devastating, especially how young they were - the youngest was 19 and I'm 19.
"It was very heartbreaking writing down the names of people who died.
"I definitely feel now people are looking at me, especially since I dyed my hair pink. It's nice to be in London and to know so many people care."
Georgina Keck, 19 from Ealing, west London, said: "To know the LGBT history here, it makes it so much more meaningful.
"Last year Pride was a celebration because of the legalisation of gay marriage but this year it'll be a funeral."
Thousands have gathered at vigils in memory of those killed in a shooting at a gay nightclub in Orlando.