The occurrence and development of colon cancer is closely related to inflammation. Therefore, this study was conducted a current retrospective research to study the effect of IL-35 (interleukin 35), a newly identified anti-infective factor, on colon cancer development. The expression of IL-35 in colon cancer samples and their adjacent normal mucosa by real-time PCR, ELISA (enzyme-linked immunosorbent assay). The effect of IL-35 on patient survival, colon cancer progression, and its effect on Wnt/β-catenine signaling pathway was also assessed. IL-35 is minimally expressed in colon cancer tissues but is highly expressed in adjacent normal tissues. The downregulation of IL-35 was significantly associated with the American Cancer Joint Committee stage and overall survival of colon cancer patients. The overexpression of IL-35 in colon cancer cells inhibits cell migration, invasion, proliferation, colony formation and cancer stem cells by inhibiting beta-catenin. IL-35 inhibits colon neoplasms in mouse. Our results suggest that IL-35 has an inhibitory effect on the development of colon cancer as a novel prognostic indicator and potential therapeutic target. © Jiang et al.