Recent research has revealed a role for Ambra1, an autophagy-related gene-related (ATG) protein, in the autophagic pro-survival response, and Ambra1 has been shown to regulate Beclin1 and Beclin1-dependent autophagy in embryonic stem cells and cancer cells. However, whether Ambra1 plays an important role in the autophagy pathway in ovarian cancer cells is unknown. In this study, we hypothesized that Ambra1 is an important regulator of autophagy and apoptosis in ovarian cancer cells. We firstly confirmed autophagic activity in ovarian cancer OVCAR-3 cells which were treated with cisplatin by assessing endogenous microtubule-associated protein 1 light chain 3 (LC3) localization and the presence of autophagosomes and LC3 protein levels in OVCAR-3 cells. Cell apoptosis and viability were measured by annexin-V and PI staining and MTT assays. We then knocked down Ambra1 expression with transfection with the plasmid expressing the small hairpin RNA (shRNA) targeting AMBRA1, then re-evaluated autophagy in the OVCAR-3 cells subject to cisplatin treatment, and re-determined the sensitivity of OVCAR-3 cells to cisplatin. Results demonstrated that cisplatin treatment induced autophagy in OVCAR-3 cells in association with Ambra1 upregulation in the ovarian cancer cells. When Ambra1 expression was reduced by shRNA, the ovarian cancer cells were more sensitive to cisplatin. In conclusion, Ambra1 is a crucial regulator of autophagy and apoptosis in ovarian cancer cells subject to cisplatin to maintain the balance between autophagy and apoptosis. And the Ambra1-targeting inhibition might be an effective method to sensitize ovarian cancer cells to chemotherapy.