Manning, 39, will officially announce his retirement in a press conference Friday.
"For 16 seasons, Eli Manning defined what it is to be a New York Giant both on and off the field," Giants president John Mara said in a statement. "Eli is our only two-time Super Bowl MVP and one of the very best players in our franchise's history. He represented our franchise as a consummate professional with dignity and accountability.
"It meant something to Eli to be the Giants quarterback, and it meant even more to us. We are beyond grateful for his contributions to our organization and look forward to celebrating his induction into the Giants Ring of Honor in the near future."
Manning steps away from the NFL holding nearly every passing record in Giants history and two Super Bowl rings. He is one of only five players in league history with at least two Super Bowl MVPs, sitting alongside Joe Montana, Tom Brady, Terry Bradshaw and Bart Starr.
His decision to retire comes after a season in which Manning spent most of his time as the backup to rookie signal-caller Daniel Jones. The Giants turned to Jones as the team's starting quarterback after Week 2 this season, and Manning started only four games, the lowest total since his rookie year.
Manning was set to become a free agent this off-season. The veteran gunslinger has earned more than $250 million from his professional football contracts, the most in league history.
The then-San Diego Chargers selected Manning with the No. 1 overall pick in the 2004 NFL Draft out of Ole Miss. He didn't want to play in San Diego and ultimately was sent to the Giants in a draft-day trade.
In four starts this season, Manning completed 61.9 percent of his passes for 1,042 yards, six touchdowns and five interceptions. He finished with a 117-117 record as a starter in the regular season and recorded 57,023 career passing yards and 366 touchdown passes.