Depression is a common complication after myocardial infarction (MI) that can seriously affect the prognosis of MI.To investigate whether formononetin could ameliorate MI injury and depressive behaviours in a mouse model of MI with depression and elucidate its underlying molecular mechanisms.Haemodynamic measurements (systolic blood pressure (SYS), the maximum rate of rise of LV pressure (± dp/dtmax)) and behavior tests (tail suspension test, sucrose preference test, forced swimming test) were used to evaluate the effects of formononetin on male C57BL/6N mice after left anterior descending (LAD) coronary artery ligation and chronic unpredictable stress. RT-qPCR, immunohistochemistry, immunofluorescence analysis, western blotting, molecular docking technology, surface plasmon resonance and gene-directed mutagenesis were used to clarify the underlying mechanism.Formononetin significantly suppressed the depressive behaviours and improved cardiac dysfunction in MI with depression mice model. Formononetin inhibited M1 polarization in macrophages/microglia, while promoting M2 polarization. Importantly, elevated serum IL-6 and IL-17A levels were found in patient with MI, and the patient serum induced M1 microglial polarization; however, formononetin reversed the polarization. Further mechanistic studies showed that formononetin inhibited GSK-3β activity and downstream Notch1 and C/EBPα signaling pathways. Covalent molecular docking showed that formononetin bound to Cys199 of GSK-3β and it has a high affinity for GSK-3β. When Cys199 was mutation, the inhibitory effect of formononetin on GSK-3β activity and M1 polarization in macrophages/microglia were also partly blocked.Our results firstly uncovered that formononetin improved cardiac function and suppressed depressive behaviours in mice after MI with depression by targeting GSK-3β to regulate macrophage/microglial polarization. More importantly, IL-6 and IL-17A produced after MI may cause neuroinflammation, which might be the key factors for depression. Formononetin may be a potential drug for treating MI with depression.Copyright © 2022. Published by Elsevier GmbH.