The spermatogenesis process is complex and delicate, and any error in any step may cause spermatogenesis arrested and even male infertility. Through mutation screening of patients with clinical azoospermia, we found a heterozygous site of the same mutation on CEP70. The centrosome protein 70 (Cep70) is involved in the regulation of microtubule assembly and shows a high expression trend during human spermatogenesis, but the specific mechanism of this protein in spermatogenesis is still unknown. Therefore, we deleted Cep70 in mice and founded that the deletion of Cep70 causes abnormal spermatogenesis and leads to male sterility. We found that knockout of Cep70 did not affect the prophase of meiosis I, but led to higher levels of thyroid hormone secretion, male germ cell apoptosis and abnormal spermiogenesis. By TEM and SEM analysis, we found that deletion of Cep70 results in abnormal formation of flagella and acrosome during spermiogenesis. TMT-labeled quantitative proteomic analysis revealed that the absence of Cep70 led to the significantly decrease of proteins associated with the formation of the flagella, head and acrosome of sperm and microtubule cytoskeleton according to the proteomic data. Taken together, our results show that Cep70 is essential for the acrosome biogenesis and flagella formation during spermiogenesis.