Introduction: Neuroinflammation and autophagy are implicated in stress -related major depressive disorder (MDD), but the underlying molecular mechanisms remain largely unknown. Objectives: Here, we identified that MDD regulated by HMGB1/STAT3/p65 axis mediated microglial activation and autophagy for the first time. Further investigations were performed to uncover the effects of this axis on MDD in vivo and in vitro . Methods: Bioinformatics analyses were used to re -analysis the transcriptome data from the dorsolateral prefrontal cortex (dlPFC) of post-mortem male MDD patients. The expression level of HMGB1 and its correlation with depression symptoms were explored in MDD clinical patients and chronic social defeat stress (CSDS)-induced depression model mice. Specific adeno-associated virus and recombinant (r) HMGB1 injection into the medial PFC (mPFC) of mice, and pharmacological inhibitors with rHMGB1 in two microglial cell lines exposed to lipopolysaccharide were used to analyze the effects of HMGB1/ STAT3/p65 axis on MDD. Results: The differential expression of genes from MDD patients implicated in microglial activation and autophagy regulated by HMGB1/STAT3/p65 axis. Serum HMGB1 level was elevated in MDD patients and positively correlated with symptom severity. CSDS not only induced depression -like states in mice, but also enhanced microglial reactivity, autophagy as well as activation of the HMGB1/STAT3/p65 axis in mPFC. The expression level of HMGB1 was mainly increased in the microglial cells of CSDS-susceptible mice, which also correlated with depressive -like behaviors. Specific HMGB1 knockdown produced a depression -resilient phenotype and suppressed the associated microglial activation and autophagy effects of CSDS-induced. The effects induced by CSDS were mimicked by exogenous administration of rHMGB1 or specific overexpression of HMGB1, while blocked by STAT3 inhibitor or p65 knockdown. In vitro , inhibition of HMGB1/STAT3/p65 axis prevented lipopolysaccharide-induced microglial activation and autophagy, while rHMGB1 reversed these changes. Conclusion: Our study established the role of microglial HMGB1/STAT3/p65 axis in mPFC in mediating microglial activation and autophagy in MDD. 2024 The Authors. Published by Elsevier B.V. on behalf of Cairo University. This is an open access article under the CC BY -NC -ND license.