The ArKO mouse cannot synthesize endogenous estrogens due to disruption of the Cyp19 gene. We have shown previously, that ArKO mice present with age progressive obesity and hepatic steatosis, and by one year of age both male and female ArKO mice develop hypercholesterolemia. In this present study, 10-12 week old ArKO mice were challenged for 90 days with high cholesterol diets. Our results show a sexually dimorphic response to estrogen deficiency in terms of cholesterol homeostasis in the liver. ArKO females presented with elevated serum cholesterol; conversely ArKO males had elevated hepatic cholesterol levels. In response to dietary cholesterol, HMG CoA reductase transcript levels were significantly reduced in females, whereas males showed more modest changes. Neither LDLR nor SREBP2 expression levels were significantly altered by diet or genotype. Expression of Cyp7a, which encodes cholesterol 7-hydroxylase, was significantly reduced in ArKO females compared with WT females, and was increased by cholesterol feeding. Cyp7a expression was significantly elevated in the WT males on high cholesterol diet although no difference was seen between genotypes on control diet. The ABCG5 and ABCG8 transporters do not appear to be regulated by estrogen. Expression of ACAT2 showed a sexually dimorphic response, where estrogen appeared to have a stimulatory effect in females but not males. This study reveals a sexual dimorphic difference in mouse hepatic cholesterol homeostasis and roles for estrogen in the regulation of cholesterol uptake, biosynthesis and catabolism in the female, but not the male.