Perfluorooctanesulfonate (PFOS) is a widespread, persistent environmental pollutant that exerts apparent liver toxicity. Flaxseed oil (FO), a dietary oil rich in α-linolenic acid, has been demonstrated to possess a diverse array of health benefits. However, whether FO protects against PFOS-induced liver injury and its underlying mechanisms remain unclear. C57/BL6 mice were orally treated with different concentrations of FO alone or in combination with 10 mg/kg of PFOS for 28 consecutive days. Blood and liver tissues were collected for proteomic, histopathological, biochemical, immunohistochemical, and molecular examinations. Results demonstrated that FO supplementation reduced PFOS-induced liver injury, as evidenced by a decrease in histopathological changes, serum transaminase (ALT and AST) levels, levels of oxidative stress, and inflammatory cytokine (TNF-α, IL-1β, and IL-6) levels. Proteomic analyses showed that differentially expressed proteins were enriched in cholesterol metabolic pathways when comparing the PFOS group to the FO supplementation groups. The expression of cholesterol metabolism-related proteins was also subsequently measured, revealing that FO supplementation decreased the protein expressions of SREBP2, HMGCR, and LDLR while increasing the expression of CYP7A1. This study demonstrates that FO can alleviate PFOS-induced hepatotoxicity by regulating hepatic cholesterol metabolism, indicating that FO may serve as an effective dietary intervention for preventing liver injury caused by PFOS.