Gunman Omar Mateen, 29, killed 50 people and wounded at least 53 others during a shooting spree at the city's popular Pulse nightclub, which caters to members of the lesbian, gay, bisexual and transgender (LGBT) community.
Spears, 34, who has long been heralded as a figurehead for the gay pride movement and has spoken out in support of LGBT civil rights organisations in the past, is deeply upset over the prejudicial killings and in a new post published on her Instagram the star urged fans to put differences aside in favour of peace.
"Music and dance take me to a wonderful, happy, safe place... And for this to happen in a place where people enjoy those things makes me so sad. This has to stop. We need to accept and love people for who they are. This is truly an attack against all of us, and I'm praying that we can join together and find a solution.