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Objective To explore whether microRNA-486-5p affected the proliferation of ovarian granulosa cells by targeting MST4 (silk/threonine protein kinase 4), thereby promoting the development of polycystic ovary syndrome (PCOS). Materials and methods The level of microRNA-486-5p in PCOS tissues and adjacent normal tissues was detected by quantitative real-time polymerase chain reaction (qRT-PCR). After microRNA-486-5p up-regulation in KNG cells, the mRNA and protein level of related genes was examined using qRT-PCR and western blot assay, respectively. Meanwhile, cell proliferation and cell cycle were analyzed by cell counting kit-8 (CCK-8) assay and flow cytometry. After insulin treatment of KNG cells, expressions of microRNA-486-5p and MST4, cell proliferation as well as cell cycle, were detected by qRT-PCR, CCK-8 and flow cytometry, respectively. Furthermore, cell proliferation and cycle situation were examined after simultaneous up-regulation of MST4 and microRNA-486-5p in vitro. Results MicroRNA-486-5p expression in PCOS tissues was significantly lower than that of normal tissues. In KNG cells, up-regulation of microRNA-486-5p significantly inhibited cell proliferation and cell cycle. The levels of cycle-associated proteins including CDK2 and CCNB1 decreased significantly. The results of dual-luciferase reporter gene assay showed that microRNA-486-5p could bind to MST4. After up-regulating microRNA-486-5p, both the mRNA and protein levels of MST4 decreased remarkably. MST4 expression was found significantly elevated in PCOS tissues as well. After overexpression of MST4, cell proliferation was enhanced, cell cycle was promoted, and expressions of cycle-related proteins increased. After treatment with different concentrations of insulin in KNG cells, the expression level of microRNA-486-5p decreased in a concentration-dependent manner. However, opposite results were observed in MST4 level. Meanwhile, the proliferation ability and cell cycle of insulin-treated cells were significantly enhanced. In addition, the inhibitory effect of microRNA-486-5p on cell proliferation and cell cycle could be partially reversed by simultaneous up-regulation of MST4 and microRNA-486-5p. Conclusions MicroRNA-486-5p can bind to MST4 in a targeted manner and inhibit the proliferation of ovarian granulosa cells, thereby inhibiting the development of PCOS.