To explore the regulator mechanisms of microRNA(miRNA) in gametogenesis of molluscs, the Zhikong scallop, Chlamys farreri, were selected, and miRNA miR-375_1, which is differentially expressed between sex during the initial sex differentiation of juvenile scallop, were targeted to conduct the present study. RT-qPCR results showed that significant expression of miR-375_1 in gonads existed during the gonadal cycle and was dynamically changed. Software prediction and dual luciferase reporter gene assay confirmed that miR-375_1 could interact with potential target genes bmi1, cfap45, dmrt1 and tssk4-1, which were closely related to spermatogenesis, and its relationship with bmi1 gene was the most significant. In situ hybridization exhibited that signals of bmi1 gene located in the spermatid and spermatozoon. The outcomes of this study indicated miR-375_1 could play important roles in spermatogenesis of C. farreri and involve in sperm differentiation and maturation through regulating the expression of bmi1gene.