Drug resistance remains a major obstacle to successful cancer treatment. Genome-wide comprehensive analysis identified a novel gene, glucocorticoid-induced protein-coding gene (DEXI), which was frequently methylated in colorectal (CRC; 36 of 73 patients; 49%) and gastric (28 of 89 patients; 31%) cancer patients. Here, we show that DEXI methylation is implicated in mechanisms facilitating resistance to camptothecin (CPT) via inhibition of apoptosis. Silencing of DEXI by siRNA significantly reduced CPT-induced apoptosis in a fibroblast cell line (1/6-fold; p<0.01) originally expressing endogenous DEXI. Restored expression of DEXI by 5-aza-2'-deoxycytidine (DAC) significantly enhanced susceptibility to CPT (3-fold; p<0.01) in a colon cancer cell line originally suppressing endogenous DEXI due to almost complete methylation. Exogenous induction of DEXI confirmed that DEXI per se contributed to enhanced susceptibility to CPT. 5-Fluorouracil (5-FU) did not exhibit these synergistic effects by DEXI restoration. Further, to estimate the clinical usefulness of DEXI methylation status as biomarker for drug resistance to irinotecan (CPT-11), 16 CRC patients who underwent FOLFIRI (5-FU + CPT-11) therapy because they were refractory to FOLFOX (5-FU + oxaliplatin) were analyzed. Significantly poor response and outcome were observed in 8 CRC patients harboring DEXI methylation. In 8 CRC patients harboring DEXI methylation disease control rate, progression-free survival and overall survival were 25.0%, 2 and 11.8 months, respectively, whereas in 8 CRC patients without DEXI methylation they were 62.5%, 5.3 and 15 months, respectively (p<0.01). These significant differences were not observed in patients undergoing treatment with FOLFOX. In conclusion, silencing of DEXI leads to resistance, but restored expression enhances susceptibility to CPT in vitro and DEXI methylation results in poor response and outcome to CPT-11-based chemotherapy, suggesting that DEXI is a potent therapeutic target and an epigenetic biomarker for the selection of patients more likely to benefit from CPT-11-based chemotherapy.