Background: In some disorders such as diabetes mellitus patients can display depressive symptoms. Metformin is among the first-line treatments for management of the type 2 diabetes mellitus which may have some anti-depressant effect. Objective: Current investigation was performed to examine the anti-depressant effects of metformin and the involvement of nitric oxide (NO) in this way, in an experimental animal model of cholestasis in NMRI (Naval Medical Research Institute) mice. Materials and Methods: Bile duct ligated (BDL) and sham-operated mice were forced to swim separately and the effect of metformin on immobility time in the last 4 minutes of the 6 minutes test was assessed. To evaluate the probable participation of NO, N-nitro-L-arginine methyl ester (L-NAME) a non-specific NO synthase inhibitor and aminoguanidine, a specific iNO synthase inhibitor were injected acutely to metformin-treated BDL mice and then their immobility time was calculated in forced swimming test (FST). Results: The immobility time significantly reduced after bile-duct ligation and metformin-treatment decreased this time additionally. L-NAME but not amino-guanidine administration significantly inhibited antidepressant like property of metformin in BDL mice. We have displayed that NO overproduction by metformin in cholestatic mice produce an anti-depressant like effect, causing a decrease in the mice immobility time in FST. Conclusion: Metformin pretreatment can decrease depression in cholestatic mice through an NO dependent pathway.