Mice lacking I-FABP (encoded by the Fabp2 gene) exhibit a gender dimorphic response to a high fat/cholesterol diet challenge characterized by hepatomegaly in male I-FABP-deficient mice. In this study, we determined if this gender-specific modification of liver mass in mice lacking I-FABP is attributable to the high fat content of the diet alone and whether hepatic Fabp1 gene (encodes L-FABP) expression contributes to this difference. Wild-type and Fabp2-/- mice of both genders were fed a diet enriched with either polyunsaturated or saturated fatty acids (PUFA or SFA, respectively) in the absence of cholesterol. Male Fabp2-/- mice, but not female Fabp2-/- mice, exhibited increased liver mass and hepatic triacylglycerol (TG) deposition as compared to corresponding wild-type mice. In wild-type mice that were fed the standard chow diet, there was no difference in the concentration of hepatic L-FABP protein between males and females although the loss of I-FABP did cause a slight reduction of hepatic L-FABP abundance in both genders. The hepatic L-FABP mRNA abundance in both male and female wild-type and Fabp2-/- mice was higher in the PUFA-fed group than in the SFA-fed group, and was correlated with L-FABP protein abundance. No correlation between hepatic L-FABP protein abundance and hepatic TG concentration was found. The results obtained demonstrate that loss of I-FABP renders male mice sensitive to high fat diet-induced fatty liver, and this effect is independent of hepatic L-FABP.