Glutathione peroxidase 4 (GPX4), a key regulator of ferroptosis, has emerged as a compelling therapeutic target for colorectal cancer (CRC). Capitalizing on the therapeutic promise of this target, we rationally designed and synthesized 24 novel Artesunate-Ebselen (ART-EBS) derivatives through structure-activity relationship (SAR) studies. Among these analogs, compound 5k exhibited the most potent antiproliferative activity against HCT116 cells with an IC₅₀ value of 0.81 ± 0.29 μM, thereby significantly outperforming its parent compounds ART (IC₅₀ > 50 μM) and EBS (IC₅₀ > 50 μM). Mechanistic investigations revealed that compound 5k triggered lipid peroxidation through the accumulation of reactive oxygen species (ROS). This was supported by multiple lines of evidence, including markedly increased ROS production, disrupted mitochondrial membrane potential, and downregulated GPX4 expression-ultimately culminating in ferroptosis of HCT116 cells. A cellular thermal shift assay (CETSA) further confirmed that compound 5k could directly bind to GPX4 protein and enhance its stability under increasing temperature, providing robust in situ evidence for the specific interaction between compound 5k and GPX4. In the CT26 xenograft model, compound 5k exerted robust tumor growth-inhibitory effects, achieving a tumor inhibition rate (TIR) of 54.9%-substantially higher than the 28.9% observed in the ART monotherapy group-without inducing significant body weight loss in mice. In contrast, all mice in the ART + compound 3k (the ebselen-based parent scaffold of compound 5k) combination group died within 24 h as a result of severe systemic toxicity. Flow cytometry analysis of immune cells in tumor-bearing mice showed that compound 5k significantly increased the proportion of CD4+ T cells in the spleen (6.2% vs. 3.5% in the control group) and promoted the infiltration of CD8+ cytotoxic T cells into tumor tissues (7.2% vs. 0.9% in the control group), indicating activation of the antitumor immune response. These findings collectively indicate that compound 5k exerts synergistic anti-CRC efficacy by triggering ferroptosis and activating antitumor immunity. Thus, compound 5k holds considerable promise as a lead ferroptosis inducer for the development of novel CRC therapeutic strategies.