Abstract: In order to investigate the anti-colon cancer activity of cucurbitacin B (CuB) and clarify the key role and molecular mechanism of ferroptosis in the anticancer effect of CuB. This study used human colon cancer cells HCT-116 as the model to evaluate the anti-colon cancer activity of CuB, detected ferroptosis-related indicators, and investigated the mechanism of CuB in inhibiting colon cancer through network pharmacology analysis, metabolomics analysis, molecular docking, and molecular dynamics simulation. The results showed that CuB significantly (P<0.05) inhibited the proliferation of HCT-116 cells with an IC₅₀ of 64.48 nmol/L. Additionally, CuB decreased the intracellular content of glutathione (GSH), promoted the accumulation of total iron ions, and enhanced the release of lactate dehydrogenase (LDH), all of which could be reversed by the ferroptosis inhibitor Fer-1. Results from network pharmacology and metabolomics analysed suggest that the mechanism of CuB's inhibition of colon cancer was linked to the GSH metabolic pathway closely related to ferroptosis. Furthermore, molecular docking revealed that CuB could bind to key proteins in the GSH metabolic pathway, SLC7A11 and GPX4, with scores of −4.819 and −3.833, respectively. Molecular dynamics simulations demonstrated that CuB had good structural stability, fluctuation, and energy stability when bound to SLC7A11. Consequently, this study uncovered that CuB exerted its anti-cancer effect by inducing ferroptosis in HCT-116 cells.