Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disorder caused by abnormal lipid metabolisms such as reduced hepatic fatty acid oxidation, but intracellular control of fatty acid oxidation under physiological and pathological conditions remains largely undefined. Here, we demonstrate that deprivation of Slc7(3) leads to hepatic steatosis in fasted zebrafish due to defects in arginine-dependent nitric oxide (NO) synthesis. Fast-induced hepatic steatosis in slc7(3) -null mutants can be rescued by treatments with NO donor, cGMP analog, AMPK activator or PPARα agonist. In contrast, inhibitors of NO synthases, AMPK or soluble guanylate cyclase and liver-specifically expressed dominant negatives of PGC-1α and PPARα are sufficient to induce hepatic steatosis in fasted wild-type larvae. Moreover, knockdown of Slc7a3 in mice or SLC7A3 in human liver cells impaired AMPK-PPARα signaling and resulted in lipid accumulation under fasting or glucose starvation, respectively. Conclusion: These findings have revealed a NO-AMPK-PPARα signaling pathway that is crucial for the control of hepatic fatty acid oxidation in vertebrates. (Hepatology 2014;).