As a first step in the transmission of malaria, Plasmodium parasites ingested by a mosquito must rapidly produce gametes prior to sexual reproduction. The phosphorylation-based signalling mechanisms underlying the initiation of gametogenesis remain largely unmapped. We have measured a high-resolution time course of phosphorylation in P. berghei gametocytes during the first minute of gametogenesis to elucidate the scope and dynamics of the response. The data reveals rapid phosphoregulation of microtubule motor proteins and DNA replication initiation proteins within the first 18s and of DNA replication machinery by 60s. The data also implicate several protein kinases and phosphatases in the gametogenesis signalling pathway. Through gene knock-out experiments, we verify that the protein kinases CDPK4 and SRPK1 have distinct influences over the phosphorylation of similar downstream targets. Together, the results show that key cell-cycle systems related to both replication and mitosis undergo simultaneous, rapid phosphoregulation.