Berberine, a natural isoquinoline alkaloid derived from Berberis species, has been reported to have anticancer effects. However, the mechanisms of action in human colorectal cancer (CRC) are not well established to date. In the present study, the cell cytotoxicity effect of berberine on human CRC cells, as well as the possible mechanisms involved, was investigated. The results of the cell viability and apoptosis assay revealed that treatment of CRC cells with berberine resulted in inhibition of cell viability and activation of cell apoptosis in a concentration‑dependent manner. To reveal the underlying mechanism of berberine‑induced anti‑tumor activity and cell apoptosis, RNA‑sequencing followed by reverse‑transcription quantitative PCR were performed. In addition, RNA immunoprecipitation, chromatin immunoprecipitation and western blot analysis were used to identify the functional regulation of CASC2/EZH2/BCL2 axis in berberine‑induced CRC cell apoptosis. The data revealed that lncRNA CASC2 was upregulated by berberine treatment. Gain‑ or loss‑of‑function assays suggested that lncRNA CASC2 was required for the berberine‑induced inhibition of cell viability and activation of cell apoptosis. Subsequently, the downstream antiapoptotic gene BCL2 was identified as a functional target of the berberine/CASC2 mechanism, as BCL2 reversed the berberine/CASC2‑induced cell cytotoxicity. lncRNA CASC2 silenced BCL2 expression by binding to the promoter region of BCL2 in an EZH2‑dependent manner. In summary, berberine may be a novel therapeutic agent for CRC and lncRNA CASC2 may serve as an important therapeutic target to improve the anticancer effect of berberine.