Oxaliplatin (Oxa) is a chemotherapy drug commonly used for advanced colorectal cancer, however most patients develop resistance after treatment while the mechanisms of which have not been fully elucidated. In this study, oxaliplatin resistant cell lines were constructed from human colorectal cancer HCT116 cells through concentration gradient induction. On this basis, we investigated the expression profiling of HCT116/Oxa cells based on quantitative proteomics. Gene ontology (GO) analysis was conducted via The Database for Annotation, Visualization, and Integrated Discovery Database (DAVID), and pathway enrichment analysis was done using GeneAnalytics database. The potential targets and molecular mechanisms of oxaliplatin resistance in colorectal cancer were further studied by inhibitors, Western blot and siRNA. The results showed that the oxaliplatin resistance index of HCT116/Oxa cells was 10.2. HCT116/Oxa cells demonstrated stronger proliferation potential and anti-apoptotic capacity to oxaliplatin compared with HCT116 cells. Proteomic data demonstrated significant expression change of 717 genes in HCT116/Oxa cells, among which 399 genes were up-regulated while 318 ones down-regulated comparing with HCT116 cells. GO enrichment analysis showed that differentially expressed genes were mainly related to biological processes such as oxidative stress response, iron metabolism, lipid metabolism, apoptosis and cell cycle progression. Pathway analysis displayed notable changes of cell metabolism, ferroptosis, Nrf2-ARE signaling, fatty acid and glutathione metabolism in HCT116/Oxa cells. Quantitative results indicated that the expression of proteins directly related to ferroptosis, including glutathione peroxidase 4 (GPX4), glutamate-cysteine ligase regulatory subunit (GCLM), ferritin light chain (FTL), ferritin heavy chain (FTH1), heme oxygenase 1 (HMOX1), glutathione reductase (GSR) and NADH dehydrogenase 1 (NQO1) increased, while long chain fatty acid-CoA ligase (ACSL) 4 and ACSL1 decreased significantly in HCT116/Oxa cells. Functional studies showed that RSL3, a specific inhibitor of GPX4, decreased the viability of drug-resistant cells, improved lipid peroxidation, increased the concentration of ferrous ions, malondialdehyde, and decreased the concentration of glutathione (GSH). Western blot showed that the expressions of GPX4, FTH1, FTL and GSR increased in HCT116/Oxa, while ACSL4 decreased. RSL3 reversed the levels of GPX4, FTH1, FTL, GSR and ACSL4. It was further found that knockdown of GPX4 decreased the viability of drug-resistant cells, increased lipid peroxidation levels and decreased GSH concentration. These results suggest that ferroptosis resistance mediated by GSH/GPX4 pathway may be a potential mechanism of oxaliplatin resistance in HCT116/Oxa, and inhibition of GSH/GPX4 signaling could be an effective approach to reverse oxaliplatin resistance in colorectal cancer.