Tumor suppressor p53 is known to play a crucial role in chemosensitivity in colorectal cancer. We previously demonstrated that an apoptosis-associated speck-like protein, ASC, is a p53-target gene which regulates p53-Bax mitochondrial apoptotic pathway. ASC is also known to be a target of methylation-induced gene silencing. An inactivation of ASC might thus cause resistance to chemotherapy, and if this is the case, then the expression of ASC would restore the chemosensitivity. The aim of this study was to clarify this hypothesis. ASC was methylated in 25% of all resected specimens in patients with colorectal cancer; however, ASC methylation did not always correspond to a lack of ASC protein. When expressed in colon cancer cells, in which ASC is absent due to methylation, ASC was found to enhance the chemosensitivity in a p53-dependent manner. In p53-null cells, ASC increased the p53-mediated cell death induced by p53-expressing adenovirus infection. Our data suggest that the methylation-induced silencing of ASC might cause resistance to p53-mediated chemosensitivity in colorectal cancer. The gene introduction of ASC may thus restore such chemosensitivity, and this modality may therefore be a useful new treatment strategy for colorectal cancer.