Hepatic steatosis is common and may progress to steatohepatitis and cirrhosis. Liver X receptor‐alpha (LXRalpha)‐sterol regulatory element binding protein‐1c (SREBP‐1c) pathway is important for hepatic steatosis. This study investigated the effect of ajoene, a stable garlic byproduct, to inhibit high fat diet (HFD)‐induced hepatic steatosis, and the molecular mechanism. Ajoene treatment attenuated fat accumulation and induction of lipogenic genes in the liver of HFD‐fed mice. Blood biochemical analyses and histopathologic examinations showed that ajoene prevented liver injury with the inhibition of oxidative stress, as evidenced by thiobarbituric acid reactive substances formation and nitrotyrosinylation. Treatment with ajoene inhibited LXRalpha agonist (T0901317)‐mediated SREBP‐1c activation, and transactivation of the lipogenic target genes in hepatocytes. Ajoene was found to activate AMP‐activated protein kinase (AMPK) through LKB1, responsible for the inhibition of p70 ribosomal S6 kinase‐1 (S6K1). The ability of ajoene to repress T0901317‐induced SREBP‐1c expression was antagonized by AMPK inhibition or S6K1 activation, proving the role of the kinases in the anti‐steatotic effect. In conclusion, ajoene has an effect to activate AMPK via LKB1 and inhibit S6K1 activity, contributing to the prevention of SREBP‐1c–mediated hepatic lipogenesis via the inhibition of LXRalpha activity.