The pathogenesis of ovarian cancer remains to be elucidated. Our previous study demonstrated that myosin heavy chain 9 (MYH9) overexpression was associated with poor prognosis of epithelial ovarian cancer. However, the mechanism of MYH9 and its regulation by microRNA (miR) is not clear. The results of the present study demonstrated that miR-6089 was one of the microRNAs targeting MYH9, and miR-6089 overexpression suppressed ovarian cancer cell proliferation, migration, invasion and metastasis in vivo and in vitro. Mechanistic studies confirmed that miR-6089 directly targeted MYH9 to inactivate the Wnt/β-catenin signalling pathway and its downstream epithelial-to-mesenchymal transition (EMT), cell-cycle factors and c-Jun, whereas overexpression of MYH9 reversed the inhibitory effects of miR-6089 overexpression in ovarian cancer cells by upregulating the Wnt/β-catenin and its downstream EMT, cell-cycle factors and c-Jun. Interestingly, miR-6089 was transcriptionally inhibited by c-Jun, a transcription factor which could be induced by MYH9 via the Wnt/β-catenin pathway. Thus miR-6089/MYH9/β-catenin/c-Jun formed a negative feedback loop in ovarian cancer. In clinical samples, miR-6089 negatively correlated with MYH9 expression. Our study is the first to demonstrate that miR-6089 serves as a tumor-suppressive miRNA, and miR-6089/MYH9/β-catenin/c-Jun negative feedback loop inhibits ovarian cancer carcinogenesis and progression.