The move was slammed by Florida state Representative Anna Eskamani, who branded it a “bigoted, transphobic piece of legislation.”
“Transgender youth deserve our love and support, not cruel attacks because of who they are,” added Ms Eskamani, a Democrat who is expected to run for Florida governor in 2022.
“The consequences for states that have passed anti-LGBT+ laws are clear - bigoted policies lead to major economic loss.
“Today Governor Ron DeSantis allowed ignorance to prevail and took our state backwards in the fight for LGBT+ equality. We won’t forget it.”
The legislation, which was approved by the Republican-led state legislature takes effect on 1 July.
“In Florida, girls are going to play girls’ sports and boys are going to play boys’ sports,” Gov. Ron DeSantis said as he signed the bill into law at a Christian academy.
“We’re going to make sure that that’s the reality.”
The law states that a transgender girl cannot take part in competitive sports without first showing a birth certificate saying she was a girl when she was born.
The Human Rights Campaign, the biggest LGBT+ group in the US, has vowed to fight the ban in court.
“On the first day of Pride, Florida Governor Ron DeSantis has signed into law a bill banning trans kids in the state from playing sports. We will be filing a lawsuit to block this arbitrary, discriminatory ban,” the group tweeted.
Human Rights Campaign President Alphonso David said the new law would harm more than just transgender girls.
“All Floridians will have to face the consequences of this anti-transgender legislation — including economic harm, expensive taxpayer-funded legal battles, and a tarnished reputation,” he added.
Observers say that the new legislation could have a severe financial impact on the state.
The NCAA, which runs university athletics in the US, has threatened to move key games and championships from any states that introduce discriminatory laws.
That could see Florida lose football bowl games and basketball tournament games, which generate millions of dollars a year for the state.
The Florida law is similar to one introduced in Idaho, which now faces major legal hurdles.
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