Spermatogenesis defects are important for male infertility; however, the etiology and pathogenesis are still unknown. Herein, we identified two loss-of-function mutations ofSTK33in seven individuals with non-obstructive azoospermia. Further functional studies of these frameshift and nonsense mutations revealed thatStk33-/KImale mice were sterile, andStk33-/KIsperm were abnormal with defects in the mitochondrial sheath, fibrous sheath, outer dense fiber, and axoneme.Stk33KI/KImale mice were subfertile and had oligoasthenozoospermia. Differential phosphoproteomic analysis andin vitrokinase assay identified novel phosphorylation substrates of STK33, fibrous sheath components A-kinase anchoring protein 3 and A-kinase anchoring protein 4, whose expression levels decreased in testis after deletion ofStk33. STK33 regulated the phosphorylation of A-kinase anchoring protein 3/4, affected the assembly of fibrous sheath in the sperm, and played an essential role in spermiogenesis and male infertility.