To explore the mechanism(s) of exercise training on ovariectomized (OVX)-induced liver lipid disorder, we observed effects of treadmill training on liver fat accumulation and ER alpha expression in intact and ovariectomized rats. Sixty female rats were randomly assigned to six groups: Sham sedentary (S-S), Sham exercised (S-EX), ovariectomized sedentary (O-S), ovariectomized exercised (O-EX), ovariectomized injected subcutaneously with 17beta-estradiol (E(2)) (O-E(2)), and ovariectomized treated with E(2) and exercise (O-E(2)-EX). Twelve weeks after intervention, OVX resulted in significantly higher body weight gain, intra-abdominal fat mass, serum levels of total cholesterol (TC), and liver triacylglycerol (TAG) concentrations and ER alpha expression than S-S group, while the relative uterus and liver mass, serum levels of E(2), TAG, and the ratio of high density lipoprotein (HDL) to TC were markedly lower in O-S group. All of these changes were decreased in O-S rats after treatment with E(2) alone with the exception of serum TC and HDL-C levels and liver ER alpha expression. Exercise alone significantly reversed the effect of OVX on serum E(2), the ratio of HDL-C to TC and the liver and intra-abdominal fat accumulation in OVX rats. The addition of E(2) to exercise induced the same uterus and lipid profile as E(2) alone. Moreover, an additive effect of exercise and E(2) was observed on liver ER alpha expression in Sham or OVX rats. In conclusion, treadmill training alone could prevent liver fat accumulation in OVX rats and the regulation of exercise on liver ER alpha expression in both OVX and Sham rats needs the presence of physical estrogen levels.