Our previous work has demonstrated that microcystin-leucine arginine (MC-LR) is a potent toxin for the reproductive system of male mice and it exerts cytotoxicity in spermatogonial cells， resulting in the constitutional and functional changes of the mouse testis. The present study was designed to investigate the functions of P-element-induced wimpy (piwi)-interacting RNAs (piRNAs) in MC-LR-induced reproductive toxicity in male mice. We observed an increase in the mmu_piR_003399 level in spermatogonial cells and mouse testicular tissues following treatment with MC-LR. Moreover， our data confirmed that cyclin-dependent kinase 6 (CDK6) was the target gene of mmu_piR_003399. Increases in the concentration of mmu_piR_003399 were correlated with the reduced expression of CDK6 both in vitro and in vivo. mmu_piR_003399 induced cell cycle arrest at the G1-phase， down-regulated sperm counts and sperm motility， and compromised sperm morphology. On the contrary， suppressing the expression of mmu_piR_003399 could substantially attenuate MC-LR-induced pathology in mice including cell cycle arrest， reduced mature sperm counts， sperm viability loss and abnormal sperm morphology. Furthermore， our data supported that mmu_piR_003399 existed in mouse serum and plasma， and its level was increased in MC-LR-treated mice. In conclusion， we demonstrate that mmu_piR_003399 plays a crucial role in regulating MC-LR-induced reproductive toxicity.