Background: The molecular mechanisms of Plasmodium invasion in mosquito midguts are not well understood. Results: The mosquito midgut peritrophic matrix protein FREP1 binds Plasmodia. Blocking parasite-FREP1 interactions or ablating FREP1 expression reduced P. falciparum infection in mosquitoes. Conclusion: FREP1 functions as a critical host factor that mediates Plasmodium invasion in mosquito midguts. Significance: Targeting FREP1 may inhibit Plasmodium transmission to mosquitoes and the spread of malaria.