Charles Silmon and Mookie Salaam also were on the winning relay team that finished in 37.58 seconds at Franklin Field to claim its third straight victory in the event.
"We wanted to make a statement today," Gatlin said. "I think we made a statement in a different way. Our first-leg guy, Charles Silmon, got bumped by the Nigerian guy that was inside but he held it together. I'm very proud of him. He still got the stick to me, I was able to get it to Mookie and Walter Dix was able to do the job and get it done."
Team USA added victories in the women's 1,600 relay, the men's distance medley relay and the women's sprint medley relay.
Oshane Bailey, a member of the winning team in the 400 relay last year in the world championships, ran a strong anchor leg for Jamaica. But Dix, who said he's finally at his best after being limited by a hamstring injury for much of the past two years, beat him to the finish by a couple of inches.
"I've been in a lot of close races," Dix said. "I can always feel if I won. I felt like I had a step on him. I knew he would basically have to throw his body across the line to win."
The U.S. had a little more breathing room in the women's 1,600, clocking in at 3:25.62 to beat second-place Nigeria and third-place Jamaica for its 13th straight Penn Relays win.
Keshia Kirtz ran the first leg in 52.7, Monica Hargrove the second in 51.7, DeeDee Trotter the third in 50.78 and Jessica Beard the anchor in 50.46.
For Trotter, a three-time Olympic medalist, the victory was a nice finishing touch on what she said was already her favorite Penn Relays.
"This year was exceptional because I had the opportunity to go into the stands yesterday and greet some of the fans - Jamaican and American - and everyone was so excited," Trotter said. "I had my medals from London and it was an exciting experience. And all of the love I showed them yesterday, I got it back on the track today. This is an atmosphere you cannot replace and is not replicated across the country."
The U.S. men's 1,600 relay team failed to claim its ninth straight Penn Relays victory when it messed up the final exchange. The Bahamas won the race in 3:00.78 with the American team of Kyle Clemons, Kind Butler, David Verburg and Manteo Mitchell second in 3:03.31.
Team USA also lost the women's 400 relay as Jamaica crossed the finish line in 42.81 to defend its title.
In the men's distance medley, Olympic medalist Leo Manzano ran a 3:57.18 mile in the anchor leg to lead the U.S. to a come-from-behind win over Australia. The American team of David Torrence, Quentin Iglehart-Summers, Brandon Johnson and Manzano finished in 9:28.7.
Ajee' Wilson's 1:59.60 anchor leg in the 800 was enough to give the Americans a win in the women's sprint medley in 3:37.94 - less than a second ahead of Jamaica.
The win was especially gratifying for Wilson, who currently attends college at nearby Temple. "Philly's become my second home," Wilson said. "It's great to be able to come here and race in my new hometown."
In the college events, Villanova won the women's 3,200 relay in 8:21.49 for its third title of the meet. Just as she did in Thursday's distance medley relay and Friday's 6,000, senior Emily Lipari anchored the Wildcats to the victory, marking the first time Villanova won three times at the Penn Relays since 1997.
Villanova also finished first in the men's 3,200, clocking in at 7:16.58.
Oregon won its second men's relay in as two days, capturing the four-mile title in 16:09.67 behind a 3:56.4 mile run from freshman sensation Edward Cheserek.
In the 1,600 relay, Texas cruised to a women's championship in 3:25.05 and Pittsburgh took the men's title in 3:03.44.
UTech (Jamaica) captured titles in the men's 800 relay (1:20.07) and the men's 400 (38.71), and Texas A&M won the women's 800 in 1:30.21.
In the individual championships, Texas A&M's Wayne Davis II won the 110 hurdles in 13.50, LSU's Jasmin Stowers took the women's 100 hurdles in 12.99, UTech's Adolphus Nevers won the men's 100 in 10.25 and Texas A&M's Olivia Ekpone won the women's 100 in 11.23.
In men's field events, Penn's Maalik Reynolds captured the high jump title on his home track with a jump of 7 feet, 2? inches. LSU's Rodney Brown took the discus at 210-9, Tennessee's Jake Blankenship won the pole vault at 17-4? and Findlay's Justin Welch won the hammer throw at 231-6.
Penn State teammates Michael Shuey and Steve Waithe picked up wins, with Shuey taking the javelin at 234-10 and Waithe the triple jump at 53-2?.