The role of gender in the progression of fatty liver due to chronic high-fat, high-fructose diet (HFFD) has not been studied. The present investigation assessed whether HFFD induced hepatic perturbations differently between genders and examined the potential mechanisms. Male, female and ovariectomized (OVX) Sprague-Dawley rats were fed either control diet or HFFD for 12 weeks. Indices of liver damage and hepatic steatosis were analyzed biochemically and histologically together with monitoring changes in hepatic gene and protein expression. HFFD induced a higher degree of hepatic steatosis in females with significant increases in proteins involved in hepatic lipogenesis. Whereas, HFFD significantly induced liver injury, inflammation, and oxidative stress only in males. Interestingly, a significant increase in hepatic fibroblast growth factor 21 (FGF21) protein expression was observed in HFFD-fed males but not in HFFD-fed females. Ovarian hormone deprivation by itself led to a significant reduction in FGF21 with hepatic steatosis, and HFFD further aggravated hepatic fat accumulation in OVX rats. Importantly, estrogen replacement restored hepatic FGF21 levels and reduced hepatic steatosis in HFFD-fed OVX rats. Collectively, our results indicate that male rats are more susceptible to HFFD-induced hepatic inflammation, and the mechanism underlying this sex dimorphism is mediated through hepatic FGF21 expression. Our findings reveal gender differences in the development of HFFD-induced fatty liver and indicate the protective role of estrogen against HFFD-induced hepatic steatosis.