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Background and Purpose: Ca2+‐activated Cl− channels (Ano1 channels) contribute to the pathogenesis of colorectal cancer. Honokiol is known to inhibit cell proliferation and tumour growth in colorectal cancer. However, the molecular target of honokiol remains unclear. This study aimed to investigate whether honokiol inhibited cell proliferation of colorectal cancer by targeting Ano1 channels. Experimental Approach Patch‐clamp techniques were performed to study the effect of honokiol on Ca2+‐activated Cl− currents in HEK293 cells overexpressing Ano1‐ or Ano2‐containing plasmids or in human colorectal carcinoma SW620 cells. Site‐directed mutagenesis was used to identify the critical residues for honokiol‐induced Ano1 inhibition. Proliferation of SW620 cells or human intestinal epithelial NCM460 cells by CCK‐8 assays. Key Results: Honokiol blocked Ano1 currents in Ano1‐overexpressing HEK293 cells and SW620 cells. Honokiol more potently inhibited Ano1 currents than Ano2 currents. Three amino acids (R429, K430 and N435) were critical for honokiol‐induced Ano1 inhibition. The R429A/K430L/N435G mutation reduced the sensitivity of Ano1 to honokiol. Honokiol inhibited SW620 cell proliferation, and this effect was reduced by Ano1‐shRNAs. Furthermore, Ano1 overexpression promoted proliferation in NCM460 cells with low Ano1 endogenous expression and resulted in an increased sensitivity to honokiol. Overexpression of the R429A/K430L/N435G mutation reduced WT Ano1‐induced increase in the sensitivity of NCM460 cells to honokiol. Conclusion and Implications: We identified a new anticancer mechanism of honokiol, through the inhibition of cell proliferation, by targeting Ano1 Ca2+‐activated Cl− channels. [ABSTRACT FROM AUTHOR]