Purpose: In the present study, we attempted to explore the role of chloride channel 3 (CLC-3) in colorectal cancer (CRC) and its related mechanism. Methods: First, the expression level of CLC-3 in CRC tumor tissues and cell lines were measured by RTqPCR, immunohistochemistry or western blot analysis. CLC-3 expression knockdown in CRC cells was achieved by siRNA transfection. The effect of CLC-3 silence on cell viability, cell cycle, invasion and migration of CRC was estimated by CCK8, flow cytometry based cell cycle assay, and transwell assay, respectively. In order to investigate whether Wnt/beta-catenin signaling was perturbed by CLC-3 knock down, CLC-3 knockdown cells were treated with pathway activator LiCl, followed by the measurement of the expressions of pathway related genes, cell viability, cell cycle, metastasis ability. Results: The expression of CLC-3 was gradually increased from normal adjacent tissues to CRC tumor tissues, and the increase in tumor tissues was related to TNM stages. CLC-3 was overexpressed in four CRC cell lines (HCT116, SW480, LoVo and SW620), compared with NCM460 cells. CLC-3 knockdown significantly reduced cell proliferation, invasion and migration ability, reflected by declined cell viability, arrested G0/G1 cell cycle, decreased invasion and migration ability. In contrast, the declined cell proliferation, invasion and migration of LoVo and SW620 cells induced by CLC-3 knockdown were reversed by the addition of Wnt/beta-catenin activator LiCl. Conclusion: CLC-3 contributed to the CRC development and metastasis through Wnt/beta-catenin signaling pathway. CLC-3 could be proposed as the candidate target for CRC treatment. (C) 2020 Elsevier Inc. All rights reserved.