Heat shock protein (Hsp) in tumor cells has been proposed to enhance their resistance to chemotherapeutic agents. In the present study, we investigated the influence of Hsp expression on the irinotecan resistance of human colorectal cancer cells. Among eight Hsp genes tested in this study, we confirmed that the expression of Hsp27 correlated with irinotecan resistance in colorectal cancer cells. Specific inhibition of Hsp27 expression using an antisense oliogodeoxynucleotide increased the irinotecan sensitivity. On the contrary, an overexpression of Hsp27 decreased the irinotecan sensitivity in colorectal cancer cells. Elevated expression of Hsp27 decreased caspase-3 activity in colorectal cancer cells. The expression level of Hsp27 determined by immunohistochemical analysis correlated with the clinical response to irinotecan in colorectal cancer patients. Hsp27 expression levels of irinotecan-non-responder (mean staining score, 6.28; proportion of high staining score, 64.2%) were significantly higher compared to those of irinotecan-responder (mean staining score, 3.16; proportion of high staining score, 33.3%) (P for t-test=0.045). These findings suggest that Hsp27 is involved in the irinotecan resistance of colorectal cancer cells possibly by reducing caspase-3 activity.