“Thanks, my Cuban friends,” the 64-year-old said in Spanish before she jumped into the water at the Hemingway International Yacht Club west of Havana at 1400 GMT.
Nyad, who donned a black and blue bathing suit, goggles and a swim cap, hopes to make the 170 kilometer (106 mile) trek in 80 hours.
Last August, Nyad broke off her fourth bid to cross the stretch after battling lightning storms and swarms of jellyfish for more than two days.
Her first attempt was in 1978, when she was 28.
Nyad set an open sea record for both men and women by swimming from the Bahamas to the Florida Keys in 1979 — a journey that is the about the same distance as the Cuba-Florida swim, but which she has described as far less dangerous.
At a news conference Friday, the veteran swimmer expressed confidence that she would make it this time around.
Not only was she hopeful that the weather and currents in the Gulf of Mexico would be better, she noted that she had a full-body suit — including a mouth guard, gloves and shoes — to protect herself from jelly fish.
She added that her dream of 35 years also sought to bring Cuba and the United States — which have been at odds for decades — closer together.
Nyad will be accompanied by a team of five yachts and two kayaks, composed of 36 people.
Australian Susan Maroney is the first and only person who has managed to swim across the Florida Straits. Protected by a shark cage, she did so in 1997 when she was 22.