Synchrony in the egress of Plasmodium ookinetes from the food bolus and enzymatic digestion of the blood meal in the mosquito midgut suggests that digestive enzymes play a role in the successful transmission of malaria parasites. Previously, we found that parasite-produced chitinase is essential for parasite transmission and can be activated by mosquito midgut protease. To determine the suitability of developing a transmission-blocking vaccine directed against mosquito trypsin-like enzyme(s), Aedes aegypti midgut trypsin-like proteases were characterized biochemically and compared to a mammalian trypsin. Mosquito trypsin is more sensitive to inhibition by aprotinin and less sensitive to egg white trypsin inhibitor than is bovine pancreatic trypsin. Soybean trypsin inhibitor and leupeptin inhibit both enzymes to similar extent. Membrane-feeding assays with aprotinin, leupeptin, and egg white trypsin inhibitor revealed a correlation between in vitro inhibition of mosquito trypsin-like activity and transmission-blocking activity. The results suggest a role for mosquito midgut trypsin(s) in malaria parasite development and indicate that the protease(s) is a potential target for blocking malaria transmission.