Haploid round spermatids undergo differentiation and morphogenesis during spermiogenesis, resulting in mature spermatozoa. The molecular details underlying this transformation, however, remain poorly understood. In this study, we generated and analyzed germ cell-specific Spata19 knockout mice (Spata19(flox/flox) ; Stra8-Cre; hereafter termed "Spata19 cKO") to assess the model that SPATA19 contributes to mitochondrial function in differentiating spermatids. Spata19-cKO males were infertile, as their sperm exhibited disorganized mitochondrial structure; furthermore, their sperm-abundance of mitochondrial proteins, activities of mitochondrial respiratory chain complex IV, and ATP levels were significantly reduced. Yet, the infertility of Spata19-cKO males was rescued by intracytoplasmic sperm injection, so the sperm are capable of initiating development. Collectively, our findings suggest that SPATA19 plays an important role in sperm motility by regulating the organization and function of the mitochondria.