Plasmodium blood stages, responsible for human to vector transmission, termed gametocytes, are the precursor cells that develop into gametes in the mosquito. Male gametogenesis works as a bottleneck for the parasite life cycle, where, during a peculiar and rapid exflagellation, a male gametocyte produces 8 intracellular axonemes that generate by budding 8 motile gametes. Understanding the molecular mechanisms of gametogenesis is key to design strategies for controlling malaria transmission. In the rodent P. berghei, the microtubule-based motor kinesin-8B (PbKIN8B) is essential for flagellum assembly during male gametogenesis and its gene disruption impacts on completion of the parasitic life cycle. In efforts to improve our knowledge about male gametogenesis, we performed an iTRAQ-based quantitative proteomic comparison of P. berghei mutants with disrupted kinesin-8B gene (ΔPbkin8B) and wild type parasites. During the 15 min of gametogenesis, ΔPbkin8B parasites exhibited important motor protein dysregulation that suggests an essential role of PbKIN8B for the correct interaction or integration of axonemal proteins within the growing axoneme. The energy metabolism of ΔPbkin8B mutants was further affected, as well as the response to stress proteins, protein synthesis, as well as chromatin organisation and DNA processes, although endomitoses seemed to occur. Significance: Malaria continues to be a global scourge, mainly in subtropical and tropical areas. The disease is caused by parasites from the Plasmodium genus. Plasmodium life cycle alternates between female Anopheles mosquitoes and vertebrate hosts through bites. Gametocytes are the parasite blood forms responsible for transmission from vertebrates to vectors. Inside the mosquito midgut, after stimulation, male and female gametocytes transform into gametes resulting in fertilization. During male gametogenesis, one gametocyte generates eight intracytoplasmic axonemes that generate, by budding, flagellated motile gametes involving a process termed exflagellation. Sexual development has a central role in ensuring malaria transmission. However, molecular data on male gametogenesis and particularly on intracytoplasmic axoneme assembly are still lacking. Since rodent malaria parasites permit the combination of in vivo and in vitro experiments and reverse genetic studies, our group investigated the molecular events in rodent P. berghei gametogenesis. The P. berghei motor ATPase kinesin-8B is proposed as an important component for male gametogenesis. We generated Pbkin8B gene-disrupted gametocytes (ΔPbkin8B) that were morphologically similar to the wild- type (WT) parasites. However, in mutants, male gametogenesis is impaired, male gametocytes are disabled in their ability to assemble axonemes and to exflagellate to release gametes, reducing fertilization drastically. Using a comparative quantitative proteomic analysis, we associated the nonfunctional axoneme of the mutants with the abnormal differential expression of proteins essential to axoneme organisation and stability. We also observed a differential dysregulation of proteins involved in protein biosynthesis and degradation, chromatin organisation and DNA processes in ΔPbkin8B parasites, although DNA condensation, mitotic spindle formation and endomitoses seem to occur. This is the first functional proteomic study of a kinesin gene-disrupted Plasmodium parasite providing new insights into Plasmodium male gametogenesis.