Enhancer of Zeste Homologue 2 (EZH2), a specific histone 3 lysine 27 (H3K27) methyltransferase, plays a critical role in tumorigenesis and cancer progression through epigenetic gene silencing and chromatin remodeling. However, the role of EZH2 in chemotherapy resistance is unknown. In this study, we found that EZH2 was overexpressed in cisplatin-resistant ovarian cancer cells compared with cisplatin-sensitive cells. Knockdown of EZH2 by RNA interference (RNAi) resensitized drug-resistant ovarian cancer A2780/DDP cells to cisplatin and decreased the level of H3K27 trimethylation (H3K27me3). Moreover, EZH2 downregulation suppressed cell proliferation and caused G2/M cell cycle arrest in A2780/DDP cells. Loss of EZH2 also enhanced sensibility of tumor xenografts to cisplatin and inhibited tumor growth in vivo. Our results indicate that EZH2 is essential for chemotherapy resistance in cisplatin-resistant cancer cells in vitro and in vivo, which is probably through H3K27 methylation as well as regulation of cell proliferation. EZH2 could be a potential novel epigenetic target to overcome drug resistance.