Reversible protein phosphorylation regulated by kinases and phosphatases controls many cellular processes. Although essential functions for the malaria parasite kinome have been reported, the roles of most protein phosphatases (PPs) during Plasmodium development are unknown. Here, we report a functional analysis of all Plasmodium berghei PPs. The Plasmodium protein phosphatome comprises 31 highly conserved PPs that exhibit differential and distinct expression patterns during various stages of the life-cycle. Systematic gene disruption analysis of all P. berghei PPs revealed that half of the genes are essential for asexual blood stage development; whereas six are essential for sexual development in the mosquito. Intriguingly, phenotypic screening coupled with transcriptome sequencing unveiled significantly altered protein kinase expression in deletion mutants of two N-myristoylated PPs. Overall, our study shows how phosphatases regulate sexual development, provides a comprehensive functional catalogue for all protein phosphatases in Plasmodium, and will inform identification of novel drug targets in malaria.