Corticosterone causes significant neurotoxicity in primary hippocampal neurons which is associated with depression. Dysfunctional autophagy is implicated in cognitive impairment and depressive-like behavior. The traditional Chinese medicine Sinisan (SNS) is highly effective in clinical treatment of depression. However, the molecular mechanisms underlying therapeutic effects of SNS are unknown. The aim of this study was to elucidate the protective effect of SNS and the underlying mechanisms against corticosterone-induced neuronal damage. The effects of serum derived from rats containing SNS (or untreated controls) on the expression of autophagy-related molecules in primary rat hippocampal neurons exposed to different concentrations of corticosterone for different intervals were explored. CCK-8 assay, LDH assay were used to analyze cell viability and LDH activity. Western blot, qRT-PCR, and immunofluorescence assays were used to determine protein and mRNA expression levels of molecules such as LC3, p62, Beclin1, ULK1, PI3K, p-PI3K, Akt p-Akt, mTOR, p-mTOR, p70S6, p-p70S6, 4ebp1 and p-4ebp1. Corticosterone induced a dose- and time-dependent reduction in cellular viability. Moreover, corticosterone (100-400 μM) treatment for 24 h increased LC3-II/LC3-I protein ratio, increased Beclin1 and ULK1 protein expression levels, and decreased p62, PI3K, p-PI3K, p-Akt, p-mTOR, p-p70S6, and p-4ebp1 protein expression levels. Notably, SNS-containing serum reversed corticosterone-induced reduction of neuronal viability, and increased p62, PI3K, p-Akt, p-mTOR, p-p70S6, and p-4ebp1 protein and mRNA expression levels. In addition, SNS-containing serum decreased LC3-II/LC3-I protein ratio, and downregulated Beclin1, and ULK1 protein and mRNA expression in primary hippocampal neurons. SNS protects primary hippocampal neurons against corticosterone-induced neurotoxicity by preventing excessive autophagy through activation of PI3K/AKT/mTOR pathway.