Surgery and chemotherapy are the gold-standard treatments for ovarian cancer. The major cause of treatment failure in patients with ovarian cancer is tumoral heterogeneity and drug resistance. Paclitaxel (PTX) is one of the most commonly used first-line drugs for ovarian cancer chemotherapy. Unfortunately, the mechanisms of PTX chemoresistance remain unclear. Here, we examined the effects of post-translational neddylation on the sensitivity of ovarian cancer cells (OCCs) to PTX-induced apoptosis. Disruption of protein neddylation with the first-in-class inhibitor MLN4924 dramatically neutralized PTX-mediated antiproliferative, antimigration, and apoptotic effects in human OCCs. Moreover, MLN4924 treatment interrupted PTX-induced microtubule polymerization. Importantly, two neddylation conjugating E2 enzymes, UBE2M and UBE2F, were found to play essential roles in PTX-induced cytotoxicity and tubulin polymerization in OCCs. In summary, our findings demonstrated that disruption of protein neddylation by MLN4924 conferred resistance to PTX and provided insights into the potential mechanisms of PTX chemoresistance in ovarian cancer.