CTRP (circumsporozoite protein and thrombospondin-related adhesive protein [TRAP]-related protein) of the rodent malaria parasite Plasmodium berghei (PbCTRP) makes up a protein family together with other apicomplexan proteins that are specifically expressed in the host-invasive stage 1. PbCTRP is produced in the mosquito-invasive, or ookinete, stage and is a protein candidate for a role in ookinete adhesion and invasion of the mosquito midgut epithelium. To demonstrate involvement of PbCTRP in the infection of the vector, we performed targeting disruption experiments with this gene. PbCTRP disruptants showed normal exflagellation rates and development into ookinetes. However, no oocyst formation was observed in the midgut after ingestion of these parasites, suggesting complete loss of their invasion ability. On the other hand, when ingested together with wild-type parasites, disruptants were able to infect mosquitoes, indicating that the PbCTRP gene of the wild-type parasite rescued infectivity of disruptants when they heterologously mated in the mosquito midgut lumen. Our results show that PbCTRP plays a crucial role in malaria infection of the mosquito midgut and suggest that similar molecular mechanisms are used by malaria parasites to invade cells in the insect vector and the mammalian host.