Metastasis is the cause of poor prognosis in ovarian cancer (OC). Enhancer of Zeste homolog 2 (EZH2), a histone-lysine N-methyltransferase enzyme, promotes OC cell migration and invasion by regulating the expression of tissue inhibitor of metalloproteinase-2 (TIMP2) and matrix metalloproteinases-9 (MMP9). Hence, we speculated that EZH2-targeting therapy might suppress OC migration and invasion. In this study, the expression of EZH2, TIMP2, and MMP9 in OC tissues and cell lines was analyzed using The Cancer Genome Atlas (TCGA) database and western blotting, respectively. The effects of SKLB-03220, an EZH2 covalent inhibitor, on OC cell migration and invasion were investigated using wound-healing assays, Transwell assays, and immunohistochemistry. TCGA database analysis confirmed that the EZH2 and MMP9 mRNA expression was significantly higher in OC tissues, whereas TIMP2 expression was significantly lower than that in normal ovarian tissues. Moreover, EZH2 negatively correlated with TIMP2 and positively correlated with MMP9 expression. In addition to the anti-tumor activity of SKLB-03220 in a PA-1 xenograft model, immunohistochemistry results showed that SKLB-03220 markedly increased the expression of TIMP2 and decreased the expression of MMP9. Additionally, wound-healing and Transwell assays showed that SKLB-03220 significantly inhibited the migration and invasion of both A2780 and PA-1 cells in a concentration-dependent manner. SKLB-03220 inhibited H3K27me3 and MMP9 expression and increased TIMP2 expression in PA-1 cells. Taken together, these results indicate that the EZH2 covalent inhibitor SKLB-03220 inhibits metastasis of OC cells by upregulating TIMP2 and downregulating MMP9, and could thus serve as a therapeutic agent for OC.© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.