The Diamond League track and field event takes place at the Stadio Olimpico on June 6 and Gatlin has been confirmed as a challenger to Bolt in the 100 metres.
Gatlin served a four-year ban from 2006 after testing positive for a banned substance - his second doping offence - and his bronze medal at the 2012 Olympics was not met with universal approval.
The meeting's technical director Luigi D'Onofrio said: "Usain Bolt's opponent in the 100 metres will be Justin Gatlin, a bronze medal winner at London 2012.
"It will be a challenge that sees two sprinters who have broken the 10-second barrier on our starting blocks.
"At least nine or 10 gold medallists are expected in Rome, diverse champions from all over the world and other athletes who stood on the podium in London."
The 33rd edition of the Golden Gala will be dedicated to the memory of Italian sprinter Pietro Mennea, who died in March at the age of 60.