"My whole motto this year is just go," the 2004 Olympic gold medalist said. "I've got Rome now and I want to go even faster."
Gatlin edged Usain Bolt on the line by 0.01 seconds last season. Bolt, the world and Olympic champion, will not be at Stadio Olimpico this year, but the likes of former world champ Kim Collins, Jamaicans Nesta Carter and Kemar Bailey Cole, and world indoor 60 champion Richard Kilty will be aiming to take Gatlin's title.
In the women's event, Shelly-Ann Fraser-Pryce will be looking to bounce back from a disappointing 200 in Eugene, where she finished last.
Fraser-Pryce is the most titled sprinter in history in the 100, with two Olympic gold medals and two world titles.
The Jamaican won the 100 in the season-opening Diamond League meet in Doha, and the race in Bellinzona on Tuesday.
"In Eugene, my body did not respond. I felt a left leg problem," Fraser-Pryce said. "I have not had a perfect start to the season but it's not a championship year. I executed the race better than in my previous 100-meter race in Doha."
Fraser-Pryce, who is looking for points to defend her Diamond Race title in the event, has never won at Stadio Olimpico and will face two athletes who have beaten her before in Rome compatriot Kerron Stewart and Murielle Ahoure.
However, Fraser-Pryce has beaten Stewart in their past 11 meetings, and Ahoure finished second to her in both the 100 and the 200 at last year's world championships in Moscow.
Also contending will be American Torie Bowie, the surprise 200 winner in Eugene.
Another highlight of the Golden Gala is the high jump, which features five men with personal bests of 2.40 meters or better.
Olympic champion Ivan Ukhov of Russia cleared a world-leading 2.41 in Doha, which was also a Diamond League record.
He faces world champion Bohdan Bondarenko of Ukraine, who jumped the same height in Moscow, where he narrowly failed to set a world record. Derek Drouin, Mutaz Essa Barshim and Aleksey Dmitrik have also cleared 2.40.
In the 100 hurdles, Australia's Olympic champion, Sally Pearson, will race world champion American Brianna Rollins, who is making her Diamond League debut, and compatriot Nia Ali, who edged out Pearson on the line in the 60 hurdles at the world indoors in March.
Attention will also be on two-time Olympic champ and world record-holder Barbora Spotakova of the Czech Republic, who will compete in the javelin for the first time since the birth of her son last year.