Chemotherapy resistance is one of the main reasons for tumor-related death. In particular, ovarian cancer patients often acquire drug resistance after chemotherapy. In this study, we found that the histone chaperone, nucleosome assembly protein 1-like 3 (NAP1L3), was significantly upregulated in tissues with cisplatin resistance compared with cisplatin-sensitive tissues. Patients with high NAP1L3 levels had poor prognosis, suggesting that NAP1L3 might regulate ovarian cancer resistance. Colony formation and terminal deoxynulceotidyl transferase nick-end-labeling (TUNEL) assays showed cells with high NAP1L3 had high cisplatin resistance, whereas cells with low NAP1L3 had poor cisplatin resistance. NAP1L3 overexpression significantly increased cisplatin resistance, whereas NAP1L3 knockdown significantly reduced cisplatin resistance, suggesting that NAP1L3 promoted cisplatin resistance. Mechanistically, gene set enrichment analysis and luciferase reporter assays showed that NAP1L3 regulated the transforming growth factor-beta (TGF-β) pathway. NAP1L3 overexpression increased the phosphorylation and nuclear translocation of SMAD family member 2 (SMAD2) and SMAD3, confirming that NAP1L3 activated the TGF-β pathway. Therefore, NAP1L3 might represent a novel target to overcome ovarian cancer chemoresistance.