AbstractPurpose: This study aimed to investigate in vivo effects of estradiol on the regulation of hepatic inducible nitric oxide synthase (iNOS) expression in the high fat (HF) diet‐induced obesity. Also, we aimed to investigate whether activation of the extracellular signal‐regulated kinase (ERK1/2), adenosine monophosphate‐activated protein kinase (AMPK), Src kinase, and miR‐221 is involved in estradiol‐mediated regulation of iNOS in the liver of obese male Wistar rats. Male Wistar rats were fed a standard laboratory diet or a HF diet for 10 weeks. Half of HF rats were treated with estradiol intraperitoneally (40 μg/kg), whereas the other half were placebo‐treated 24 H before euthanasia. Results show that estradiol treatment of HF rats decreased hepatic iNOS mRNA (P < 0.05) and protein expression (P < 0.01), the protein levels of p65 subunit of nuclear factor κB (P < 0.05) and ERα (P < 0.05), ERK1/2 phosphorylation (P < 0.001), and ERα/Src kinase association (P < 0.05). By contrast, hepatic Src protein level (P < 0.05), AMPKα phosphorylation (P < 0.05), and miR‐221 expression (P < 0.05) were increased in HF rats after estradiol treatment. Our results indicate that estradiol in vivo regulates hepatic iNOS expression in obese rats via molecular mechanisms involving ERK1/2, AMPK, Src, and miR‐221 signaling.