Dysfunction of the DNA repair pathway contributes to tumorigenesis and drug resistance. Methyl methanesulfonate and ultraviolet sensitive gene clone 81 (MUS81), a key endonuclease in DNA repair, is generally considered a tumor suppressor; however, recent studies have revealed its tumor-promoting effect in epithelial ovarian cancer (EOC) and have shown that its overexpression is associated with cisplatin sensitization. However, the exact functional role of MUS81 and its regulation in relation to chemotherapy sensitivity remains unknown. Our previous study using protein interaction chip revealed that minichromosome maintenance complex component 2 (MCM2) is closely correlated with MUS81. This study aimed to investigate the biological effects and mechanisms of MUS81 on cellular responses to chemotherapeutic drugs. To accomplish this, we downregulated MUS81 and MCM2 in A2780 and SKOV3 ovarian cancer cells using lentivirus-mediated RNAi. Using a qPCR-based HR assay kit to detect HR efficiency. The sensitivity of MUS81 to olaparib was investigated by cell proliferation, colony formation assays and flow cytometry. The results showed that MUS81 modulates MCM2 levels as well as homologous recombination (HR) activity. Moreover, downregulation of MUS81 increased the sensitivity of EOC cells to olaparib by inducing S phase arrest and promoting apoptosis through activation of MCM2. MUS81 may be a potential novel therapeutic target for EOC.