Polamalu, a former USC fullback, coached running backs and held the title of offensive coordinator for the last three seasons.
The embattled Kiffin, however, was the play-caller and ran the offense for a team that opened the season No. 1 but finished 7-6, despite having senior quarterback Matt Barkley, All-American receivers Robert Woods and Marqise Lee and running backs Silas Redd and Curtis McNeal, both of whom rushed for more than 1,000 yards the previous season.
USCfootball.com, citing "multiple sources," first reported that Polamalu would not return next season.
Polamalu, the only full-time assistant who had played for the Trojans, did not return phone messages. A USC athletic department spokesman declined comment.
Polamalu's departure comes two days after USC signed a recruiting class that features running backs Justin Davis and Ty Isaac.
Several current and former USC players expressed their feelings about Polamalu through social media.
"Not only did we lose the best coach, we lost the only Trojan who was on staff. Real sad day," fullback Soma Vainuku posted on Twitter.
Polamalu is the fourth full-time coach, the first from the offense, to depart since the Trojans' embarrassing 21-7 loss to Georgia Tech in the Sun Bowl.
Monte Kiffin, former assistant head coach for defense, resigned and is now the Dallas Cowboys defensive coordinator. Linebackers coach Scottie Hazelton is defensive coordinator at Nevada and secondary coach Marvin Sanders was fired.
New defensive coordinator Clancy Pendergast will oversee defensive backs.
A few days before the Sun Bowl, which the Trojans lost to Georgia Tech on Dec. 31, Polamalu said he loved coaching at USC and had no plans to leave.
"Until someone says, 'Hey, I don't want you here, you know, then I'll maybe go and work on the academic side of the building," he said. "It's going to take a lot because this is where I want to be."
Kiffin gave Polamalu and quarterbacks coach Clay Helton more offensive responsibility in the run-up to the Sun Bowl. Polamalu said "every coach would love" to call plays, but did not sound optimistic that Kiffin would relinquish play-calling in the 2013 season.
"I have a little personality flaw," he said of his offensive philosophy. "I want to be real physical."
Polamalu was in his second coaching stint with the Trojans. He coached running backs and special teams from 2000 to 2003 under Paul Hackett and Pete Carroll before coaching running backs in the NFL with the Cleveland Browns and Jacksonville Jaguars.
In 2010, Polamalu was set to begin his first season with the Tennessee Titans when Kiffin hired him, a move that drew criticism from then-Titans coach Jeff Fisher and spurred the Titans to sue USC and Kiffin. The NFL team and USC later announced they had reached a "mutually satisfying arrangement" to end the lawsuit.