Transmission from the vertebrate host to the mosquito vector represents a major population bottleneck in the malaria life cycle that can successfully be targeted by intervention strategies. However, to date only about 25 parasite proteins expressed during this critical phase have been functionally analysed by gene disruption. We describe the first systematic, larger scale generation and phenotypic analysis of Plasmodium berghei knockout (KO) lines, characterizing 20 genes encoding putatively secreted proteins expressed by the ookinete, the parasite stage responsible for invasion of the mosquito midgut. Of 12 KO lines that were generated, six showed significant reductions in parasite numbers during development in the mosquito, resulting in a block in transmission of five KOs. While expression data, time point of essential function and mutant phenotype correlate well in three KOs defective in midgut invasion, in three KOs that fail at sporulation, maternal inheritance of the mutant phenotype suggests that essential function occurs during ookinete formation and thus precedes morphological abnormalities by several days.