Jamesville-Dewitt ends Spencerport's 64-match unbeaten streak.
It's the first time the Rangers have lost since 2015.
Hayley Quackenbush scored off a corner kick in the 94th minute to give Jamesville-DeWitt a 1-0 win over the two-time state champions on Saturday night.
J-D will play Valley Stream South on Sunday at 12:30 pm.