Schlafen-11 (SLFN11) showed a highly significant positive correlation with the response of topoisomerase inhibitors in cancer cell lines derived from prostate, lung, etc. However, this finding has not been validated in colorectal cancers (CRCs). Although irinotecan (CPT-11), a topoisomerase inhibitor, is one of the most important drugs in the treatment of advanced and/or metastatic CRC, resistance is a critical drawback to its clinical effectiveness. The present study aimed to investigate the mechanism of SLFN11 in the response of CRC cell lines to SN-38 (an active CPT-11 metabolite) treatment. Western blotting was used to measure protein expression levels of SLFN11 in human CRC cell lines. Then, SLFN11 expression was modulated by transfecting human CRC cell lines with vectors carrying the SLFN11 gene or specific SLFN11 small interfering RNAs. The effects of SN-38 treatment on CRC cells with different SLFN11 expression levels were detected, including inhibition of cell growth, induction of apoptosis, and cell cycle arrest. This study showed that SLFN11 expression varied between the CRC cell lines and high-level SLFN11 expression promoted SN-38-induced antiproliferative activity, apoptosis, and cell cycle arrest. Our results suggest that SLFN11 plays a key role in cell cycle arrest and/or induction of apoptosis in response to exogenous SN-38-induced DNA damage and might be used as a new predictive biomarker for CRC treatment.