Objective This study aims to systematically elucidate the molecular mechanism of reserpine-induced depression through network toxicology, molecular docking techniques, behavioral assessments of animal models, and histopathological analyses. Methods Core targets were screened using multi-database network toxicology, followed by constructing a protein-protein interaction (PPI) network and validating core targets through molecular docking. Sprague-Dawley (SD) rats were randomly divided into a control group and a reserpine-treated group (0.5 mg·kg?1), receiving corresponding treatments once daily for 4 consecutive days. Behavioral changes were assessed using the forced swim test and open field test. Serum neurotransmitters were quantified by enzyme-linked immunosorbent assay (ELISA). Neuropathological damage was observed via tissue staining. Target gene expression regulation was verified through Western blotting. Results Network toxicology screening and molecular docking simulation results demonstrated that reserpine exhibited significant binding affinity with dopamine D2 receptor (DRD2), cyclic-AMP response element binding protein (CREB), and serine/threonine-protein kinase 1 (AKT1). Animal experiments demonstrated that the treated group displayed depression-like behaviors including motor inhibition (P<0.05), with decreased serum levels of norepinephrine (NE) and 5-hydroxytryptamine (5-HT) (P<0.05，P<0.05). Pathological observations revealed microglial proliferation in the cerebral cortex, increased apoptosis, and reduced Nissl bodies in the hippocampal CA1 region. Downregulation of brain-derived neurotrophic factor (BDNF) in brain tissue, along with decreased expression of hippocampal AKT1 and phosphorylated AKT1 (p-AKT1), was also observed. Conclusion Reserpine has been demonstrated to exert an influence on monoamine transmitter metabolism and neuronal structural integrity through the inhibition of BDNF and AKT1 protein expression, which can result in the manifestation of depressive-like behavior and cerebral nerve damage in rats.