Pumpkin seed oil (PSO) possesses multiple pharmacological properties, including antioxidant, anti-inflammatory, and anti-apoptotic effects. Given that paracetamol (PCM) is a common cause of drug-induced hepatic injury, the present study aimed to evaluate the hepatoprotective potential of PSO against PCM-induced liver toxicity in rats and to elucidate its underlying mechanisms. Male albino rats were allocated into six groups (n = 6): control, PCM, silymarin (SLM, 50 mg/kg/day), PSO (1.5 mg/kg/day), SLM + PCM, and PSO + PCM. Serum and liver samples were examined for biochemical, molecular, and histopathological changes. GC–MS analysis identified six major fatty acid methyl esters in PSO, which may contribute to its biological activity. PSO significantly mitigated PCM-induced hepatotoxicity by restoring liver function markers, enhancing antioxidant defenses via downregulation of CYP2E1 and activation of Nrf2, and attenuating inflammation through suppression of TNF-α and IL-1β while elevating IL-10 levels. Moreover, PSO reduced TGF-β expression and improved hepatic regeneration. Histopathological evaluation confirmed the protective effects, and modulation of BAX/Bcl-2 balance indicated its anti-apoptotic action. In conclusion, PSO exerts potent hepatoprotective effects against PCM-induced liver injury, likely mediated by its bioactive constituents through modulation of CYP2E1 and Nrf2 signaling pathways. These findings highlight PSO as a promising natural candidate for further preclinical and clinical evaluation in hepatoprotection.