Objective: Recent studies have indicated the possibility that genistein may improve depression via regulating the expression of miR-221/222. This study is to explore whether genistein could improve depression by altering miR-221/222 levels and investigate the possible mechanisms involved in the improvement effect of genistein. Methods: The animal model of depression was established through unpredictable chronic mild stress. Nest building test and splash test were adapted to evaluate the effects of genistein on depressive symptoms in mice. qRT-PCR and western blot analysis were used to detect the expression of miR-221/222 and connexin 43 (Cx43) in the prefrontal cortex of the mice. In vitro, U87-MG astrocytes were treated with genistein and the expression of miR-221/222 and Cx43 was measured. The dual-luciferase reporter assay was used to verify whether Cx43 was a direct target of miR-221/222. Results: The behavioral tests showed that genistein could significantly reduce depression symptoms of mice, and this remission was not affected by gender. Genistein in vivo and in vitro could reduce increased levels of miR-221 and miR-222 in the prefrontal cortex of depressed mice, while upregulate Cx43 expression. Dual-luciferase reporter assay suggested Cx43 was directly regulated by miR-221/222 in astrocytes. Conclusion: Genistein can play its antidepressant effect through down-regulating miR-221/222 by targeting Cx43.