Abstract: This study aimed to investigate the major metabolites of Tolypocladium sinense fermentation broth (TSFB) and evaluate its in vitro antioxidant activity, as well as its ability to alleviate hydrogen peroxide (H₂O₂)-induced oxidative stress in mouse hippocampal neuronal cells (HT22). Ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) was employed for untargeted metabolomic analysis of TSFB. An antioxidant evaluation system combined with Western blotting was used to assess the antioxidant effects of TSFB and its influence on the expression levels of superoxide dismutase 1 (SOD1), nuclear factor erythroid 2-related factor 2 (Nrf-2), superoxide dismutase 2 (SOD2), and heme oxygenase-1 (HO-1). The results revealed that detected 2745 metabolites from TSFB, primarily composed of lipids and lipid-like molecules, organic acids and derivatives, and organoheterocyclic compounds. The dominant bioactive constituents included sphingosine, citric acid, and phytosphingosine, which exhibited the highest relative peak areas. In vitro antioxidant assays demonstrated that TSFB possessed strong free radical scavenging capacity. In the oxidative stress cell model, the results showed that both low and high doses of TSFB treatment significantly increased cell survival rates (P<0.001). Meanwhile, oxidative stress markers such as MDA content, LDH levels, and ROS levels were significantly reduced (P<0.05), while the antioxidant capacity indicator GSH-Px levels were significantly elevated (P<0.05). Additionally, the high-dose TSFB group notably increased SOD levels (P<0.01). Both low- and high-dose TSFB treatment groups significantly upregulated the protein expression of Nrf-2, HO-1, and SOD1 (P<0.05), and the high-dose group further significantly increased SOD2 protein expression (P<0.001). This study systematically analyzed the major metabolites and bioactive components of TSFB, elucidating its antioxidant mechanisms and highlighting its potential therapeutic applications.