Our previous studies have indicated that osthole may ameliorate the hepatic lipid metabolism and inflammatory response in nonalcoholic steatohepatitic rats, but the underlying mechanisms remain unclear. This study aimed to determine whether the effects of osthole were mediated by the activation of hepatic peroxisome proliferator-activated receptor α/γ (PPARα/γ). A rat model with steatohepatitis was induced by orally feeding high-fat and high-sucrose emulsion for 6 weeks. These experimental rats were then treated with osthole (20 mg/kg), PPARα antagonist MK886 (1 mg/kg) plus osthole (20 mg/kg), PPARγ antagonist GW9662 (1 mg/kg) plus osthole (20 mg/kg) and MK886 (1 mg/kg) plus GW9662 (1 mg/kg) plus osthole (20 mg/kg) for 4 weeks. The results showed that after osthole treatment, the hepatic triglycerides, free fatty acids, tumor necrosis factor-α, monocyte chemotactic protein-1, interleukin-6 (IL-6), IL-8 and liver index decreased by 52.3, 31.0, 32.4, 28.9, 36.3, 29.3 and 29.9%, respectively, and the score of steatohepatitis also decreased by 70.0%, indicating that osthole improved the hepatic steatosis and inflammation. However, these effects of osthole were reduced or abrogated after simultaneous addition of the specific PPARα antagonist MK886 or/and the PPARγ antagonist GW9662, especially in the co-PPARα/γ antagonists-treated group. Importantly, the osthole-induced hepatic expressions of PPARα/γ proteins were decreased, and the osthole-regulated hepatic expressions of lipogenic and inflammatory gene proteins were also reversed by PPARα/γ antagonist treatment. These findings demonstrated that the ameliorative effect of osthole on nonalcoholic steatohepatitis was mediated by PPARα/γ activation, and osthole might be a natural dual PPARα/γ activator.