Although simple steatosis was originally thought to be a pathologically inert histological change, fat accumulation in the liver may play a critical role not only in disease initiation, but also in the progression to nonalcoholic steatohepatitis (NASH) and cirrhosis. Therefore, prevention of fat accumulation in the liver may be an effective therapy for multiple stages of NAFLD. Promising beneficial effects of betaine supplementation on human NAFLD have been reported in some pilot clinical studies; however, data related to betaine therapy in NAFLD are limited. In this study, we examined the effects of betaine on fat accumulation in the liver induced by high-sucrose diet and evaluated mechanisms by which betaine could attenuate/prevent hepatic steatosis in this model. Male C57BL/6 mice weighing 20 ± 0.5 g (mean ± SEM) were divided into four groups (8 mice per group) and started on one of 4 treatments: standard diet (SD), SD+betaine, high-sucrose diet (HS) and HS + betaine. Betaine was supplemented in the drinking water at a concentration of 1% (w/v) (anhydrous). Long-term feeding of high-sucrose diet to mice caused significant hepatic steatosis accompanied by markedly increased lipogenic activity. Betaine significantly attenuated hepatic steatosis in this animal model, and this change was associated with increased activation of hepatic AMP-activated protein kinase (AMPK) and attenuated lipogenic capability (enzyme activities and gene expression) in the liver. Our findings are the first to suggest that betaine might serve as a therapeutic tool to attenuate hepatic steatosis by targeting the hepatic AMPK system.