Scallops belong to bivalves of the mollusc phylum and are important shellfish culture species in China. Important varieties of scallops and their complex reproductive regulation mechanisms have been the focus of research in shellfish biology. Many genes have been reported to be involved in the sex differentiation and gonadal development of scallops, whereas the regulation of these genes has rarely been reported. MicroRNA is a class of important endogenous regulatory factors that can participate in the regulation of gene expression. To explore the role of microRNAs in the gonadal development of shellfish, this study took the Zhikong scallop (Chlamys farreri), a hermaphroditic and sex-stable bivalve species and thus good material for such a mechanism exploration, as the research material. In a previous study, we found that miR-124-3p_4 was highly expressed in the testes of juveniles during the early stage of sexual differentiation, and thus took this microRNA as the research object. The sequence of miR-124-3p_4 was compared with other species and the core sequence was consistent. The genes targeted by miR-124-3p_4 were predicted through RNAhybrid and miRanda, from which 729 and 436 target genes were obtained, respectively, and the intersection was 260. Combining with the transcriptome data of C. farreri, eight ovary bias genes and twelve testis genes were obtained. Based on the binding site and binding-free energy analysis, all eight ovary bias genes matched perfectly with miR-124-3p_4 and their free energy was all below –20 kcal/mol, indicating the interaction activities between them. Among these genes, dpgn-like was found to have the strongest interaction with miR-124-3p_4. Sequence and structure analyses of the dpgn-like gene showed that its cDNA full length was 4526 bp, coding 559 amino acids. Four MFS, two KAZAL_FS, and one OATP conserved domain were identified in the DPGNL protein. Phylogenetic results clarified its identity and evolutionary status. The expression of the dpgn-like gene was mainly in the mantle, foot, gill and gonads, of which the expression in the ovaries was significantly higher than that in the testis with a fold change of 4.4. In situ hybridization was performed in mature ovaries and results showed that the dpgn-like gene expression was localized in oogonia and oocyte cytoplasm, but not in follicle cells, indicating its potential involvement in ovary development. To test whether miR-124-3p_4 and the dpgn-like gene were directly interacted, a dual luciferase reporter gene analysis was conducted. Through co-transfer of miR-124-3p_4 and the dpgn-like gene into HEK293T cells, the dpgn-like gene expression was significantly reduced by 64%. Furthermore, in vivo overexpression of miR-124-3p_4 in the ovary was explored by the microinjection of miR-124-3p_4 agomir. After 3 d of microRNA overexpression, a 16.28-fold increase of miR-124-3p_4 to the control group was confirmed by RT-qPCR. Meanwhile, the dpgn-like gene expression was reduced to 0.74, again indicating that miR-124-3p_4 could combine and down-regulate dpgn-like gene expression in ovaries. In summary, this study screened for the potential target genes of miR-124-3p_4, identified the dpgn-like gene which had the strongest interaction with it, revealed the sequence and structure characteristic of the dpgn-like gene, and uncovered its expressional patterns. These results suggest that the dpgn-like gene may be involved in gonadal development. In addition, both in vitro and in vivo analyses indicated that miR-124-3p_4 could directly target the dpgn-like gene. Collectively, these outcomes reveal that male-biased miR-124-3p_4 negatively regulated ovary-biased dpgn-like gene expression in the testis, implying its potential role during the development of gonads.