The aim of this study is to explore the function of USP14 on the sensitivity of retinoblastoma (RB) to cisplatin (DDP) and the underlying mechanism. USP14 was knockdown in Y79 cells by transfecting three siRNAs (si-USP14-1, si-USP14-2, and si-USP14-3), with si-USP14 NC as the negative control. si-USP14-3 was selected by results of Western blotting. The CCK-8 assay was used to detect the IC50 of Y79 cells and the growth curve. The cell cycle, cell apoptosis, and ROS level were measured by flow cytometry. The expression level of P-GP, ERCC1, survivin, GPX4, FTH1, ACSL4, NOX1, COX2, and FASN was determined by the Western blotting assay. CO-IP assay was utilized to evaluate the interaction between USP14 and FASN. The IC50 of DDP in Y79 cells and Y79/DDP cells was 7.83 µM and 24.67 µM, respectively. Compared to control and si-USP14 NC groups, increased apoptotic rate and ROS level, and arrested cell cycle in S phase were observed in USP14-knockdown Y79 cells. Compared to control and si-USP14 NC groups, increased apoptotic rate and arrested cell cycle in G0/G1 phase were observed in USP14-knockdown Y79/DDP cells. Compared to control, increased ROS level was observed in USP14-knockdown Y79/DDP cells. Compared to the si-USP14 NC groups, extremely downregulated P-GP, ERCC1, survivin, GPX4, FTH1, NOX1, COX2, and FASN were observed in USP14-knockdown Y79 cells or Y79/DDP cells, accompanied by the elevated expression of ACSL4. The interaction between USP14 and FASN was identified according to the result of CO-IP assay. By silencing USP14 in Y79 and Y79/DDP cells, levels of resistance-related proteins (P-GP, ERCC1, and survivin), ferroptosis-related proteins (FTH1 and GPX4), and lipid metabolism-related proteins (NOX1, COX2, and FASN) were dramatically reduced, accompanied by enhanced ROS level, increased apoptosis, and restrained DNA content, indicating that USP14 might suppress the DDP resistance in RB by mediating ferroptosis, which is an important target for treating RB.