BACKGROUND AND PURPOSE Mistletoe lectin‐I (ML‐I), the main anti‐cancer component of mistletoe extracts, was originally thought to act exclusively on 28S rRNA. Here, we investigate the down‐regulating effect and mechanism of CM‐1, an ML‐I isolated from Chinese mistletoe, on some miRNAs. EXPERIMENTAL APPROACH The anti‐cancer effects of CM‐1 were assessed in vitro and in vivo in colorectal cancer cells. The miRNAs down‐regulated by CM‐1 were identified by miRNA microarray assay and validated by qRT‐PCR analysis. The suppression of host gene transcription or by degradation of precursors was determined by qRT‐PCR and enzyme activity assays respectively. The qRT‐PCR, Western blot and immunohistochemistry were used to examine the expression of their target gene and related downstream effector. Cell proliferation was assayed in stably transfected HEK‐293 cells with different levels of these miRNAs. KEY RESULTS CM‐1 showed prominent anti‐neoplastic activity towards CLY and HT‐29 cells both in vitro and in vivo. The miR‐135a&b were the miRNAs most down‐regulated by CM‐1. Their host gene transcription was largely up‐regulated, while their precursors were degraded directly by CM‐1. The expression of their target gene adenomatous polyposis coli and the phosphorylation of related effector β‐catenin were both significantly up‐regulated. The IC50 values of CM‐1 on derivative HEK‐293 cells with high miR‐135a&b levels were 2–4 times lower than that of control cells. CONCLUSIONS AND IMPLICATIONS CM‐1 down‐regulated some miRNAs by degrading their precursors, which contributes to its prominent anti‐cancer activity. LINKED ARTICLE This article is commented on by Rushworth, pp. 346–348 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2010.01075.x