Colorectal cancer is a major global health burden, with limited efficacy of traditional treatment modalities in improving survival rates. However, recently advances in immunotherapy has improved treatment outcomes for patients with this cancer. To address the continuing need for improved treatment efficacy, this study introduced a novel tri-specific antibody, IMT030122, that targets EpCAM, 4-1BB, and CD3. We evaluated the pharmacological efficacy and mechanism of action of IMT030122 in vitro and in vivo. In in vitro studies, IMT030122 exhibited differential binding to antigens and cells expressing EpCAM, 4-1BB, and CD3. Moreover, IMT030122 relied on EpCAM-targeted activation of intracellular CD3 and 4-1BB signaling and mediated T cell cytotoxicity specific to HCT116 colorectal cancer cells. In vivo, IMT030122 demonstrated potent anti-tumor activity, significantly inhibiting the growth of colon cancer HCT116 and MC38-hEpCAM subcutaneous grafts. Further pharmacological analysis revealed that IMT030122 recruited lymphocytes from peripheral blood into colorectal cancer tissue and exerted durable anti-tumor activity, predominantly by promoting the activation, proliferation, and differentiation of CD8T cells. Notably, IMT030122 still exhibited anti-tumor efficacy even in the presence of significantly depleted lymphocytes in colorectal cancer tissue. The potent pharmacological activity and anti-tumor effects of IMT030122 suggest it may enhance treatment efficacy and substantially extend the survival of patients with colorectal cancer in the future.Colorectal cancer is a major global health burden, with limited efficacy of traditional treatment modalities in improving survival rates. However, recently advances in immunotherapy has improved treatment outcomes for patients with this cancer. To address the continuing need for improved treatment efficacy, this study introduced a novel tri-specific antibody, IMT030122, that targets EpCAM, 4-1BB, and CD3. We evaluated the pharmacological efficacy and mechanism of action of IMT030122 in vitro and in vivo. In in vitro studies, IMT030122 exhibited differential binding to antigens and cells expressing EpCAM, 4-1BB, and CD3. Moreover, IMT030122 relied on EpCAM-targeted activation of intracellular CD3 and 4-1BB signaling and mediated T cell cytotoxicity specific to HCT116 colorectal cancer cells. In vivo, IMT030122 demonstrated potent anti-tumor activity, significantly inhibiting the growth of colon cancer HCT116 and MC38-hEpCAM subcutaneous grafts. Further pharmacological analysis revealed that IMT030122 recruited lymphocytes from peripheral blood into colorectal cancer tissue and exerted durable anti-tumor activity, predominantly by promoting the activation, proliferation, and differentiation of CD8T cells. Notably, IMT030122 still exhibited anti-tumor efficacy even in the presence of significantly depleted lymphocytes in colorectal cancer tissue. The potent pharmacological activity and anti-tumor effects of IMT030122 suggest it may enhance treatment efficacy and substantially extend the survival of patients with colorectal cancer in the future.