Male aromatase knockout mice (ArKO; an estrogen-deficient model) present with male-specific hepatic steatosis that is reversible upon 17beta-estradiol replacement. This study aims to elucidate which estrogen receptor subtype, ERalpha or ERbeta, is involved in the regulation of triglyceride homeostasis in the liver. Nine month-old male ArKO mice were treated with vehicle, ERalpha- or ERbeta-specific agonists via subcutaneous injection, daily for 6 weeks. Male ArKO mice treated with ERalpha agonist had normal liver histology and triglyceride contents compared to vehicle-treated ArKO; omental (gonadal) and infra-renal (visceral) fat pad weights were normalized to those of vehicle-treated WT. In contrast, ERbeta agonist-treatment did not result in the similar reversal of these ArKO phenotypes. In vehicle-treated ArKO mice, hepatic transcript expression of Fasn and Scd1 (key enzymes in de novo FA synthesis) were significantly elevated as compared to vehicle-treated WT, but only Fasn expression was lowered to WT level after ERalpha agonist-treatment. There were no significant changes in the transcript levels of Cpt1 (required for transfer of FA residues into the mitochondria for beta-oxidation) and Srebf1c (the upstream regulator of de novo FA synthesis). We also confirmed by RT-PCR that only ERalpha is expressed in the mouse liver. There were no changes in hepatic androgen receptor transcript level across all treatment groups. Our data suggest that estrogens act via ERalpha to regulate triglyceride homeostasis in the ArKO liver. Since the liver, adipose tissue and arcuate nucleus express mainly ERalpha, estrogens could regulate hepatic functions via peripheral and central pathways.