<p>Colorectal cancer (CRC) represents one of the most prevalent forms of malignant neoplasms affecting the digestive tract. Recent studies have demonstrated that the induction of ferroptosis in tumor cells represents a novel therapeutic strategy. Ononin, an isoflavone glycoside compound derived from traditional Chinese medicinal plants, has garnered attention for its purported therapeutic efficacy. This study integrated network pharmacology and experimental studies to elucidate the underlying mechanism involved in the therapeutic action of ononin against CRC. The results demonstrated that ononin induced lipid peroxidation and a reduction in mitochondrial membrane potential (MMP), indicative of mitochondrial damage in CRC cells, accompanied by a pronounced decline in GPX4 expression. The combination of ononin and anti-PD-L1 therapy demonstrated a notable enhancement in tumor growth inhibition in the MC38 mouse CRC model, accompanied by a marked increase in the proportion of intratumoral IFNγ⁺CD8⁺T cells. In summary, ononin triggers ferroptosis in colorectal cancer cells by increasing lipid ROS through the PI3K/AKT/Nrf2 pathway and reducing GPX4 expression. The combination of ononin and anti-PD-L1 therapy shows a synergistic anti-tumor effect, effectively inhibiting tumor growth and enhancing immune response. This study demonstrates that ononin is a promising therapeutic agent for CRC and may potentially serve as an immuno-adjuvant to enhance immunotherapy efficacy.</p>