Cancer stem cells (CSCs) function as the driving force of cancer initiation and progression. Wnt/β-catenin is the core pluripotency pathway in CSCs, while its crucial regulator has not been fully elucidated yet. Here, we evaluated the role of ZO-1, a component of the tight junction protein complex, in colorectal CSCs, and found ZO-1 downregulation in both colorectal cancer cells and spheres. Over-expression of ZO-1 can inhibit the sphere-forming capacity and CSC marker expression in spheres. Immunofluorescence staining and co-immunoprecipitation analysis further revealed the interaction between ZO-1 and β-catenin and the repressed role of ZO-1 in β-catenin nuclear accumulation. Using in vitro and in vivo models, we suggested the suppression effects of sulforaphane on CSCs via the ZO-1/β-catenin axis in colorectal cancer. The findings from this study depicted for the first time that ZO-1 dampened colorectal CSCs by interacting with β-catenin and attenuated its nuclear translocation, providing new insights into the mechanisms and applications of sulforaphane in targeting CSCs.