This study was conducted to evaluate the efficacy and safety of the combination of mitomycin-C (MMC) and S-1 as third-line chemotherapy for patients with advanced colorectal cancer (CRC) showing resistance to irinotecan- and oxaliplatin-containing regimens. Patients were recruited into the study from January 2009 and 10 patients were enrolled for 10 months. However, since no patients had shown a response by 10 months, the study was terminated early according to the protocol. MMC 7 mg/m(2) was administered intravenously on day 1 every 6 weeks in the first 4 cycles. S-1 was administered twice daily at 35 mg/m(2), within 1 h of meals on days 1-14. Following a rest for 7 days, S-1 was administered again on days 22-35, followed by a 7-day rest. A total of 14 cycles were delivered for 10 patients. All 10 patients were assessable for response. A total of 3 patients (30%) had stable disease and the remaining 7 showed disease progression. With a median follow-up of 7 months, the median overall survival was 10.5 months. Grade 3-4 myelotoxicities included neutropenia in two patients, anemia in two and thrombocytopenia in one. Grade 1-2 nausea and vomiting developed in 5 patients. One patient experienced grade 3 diarrhea. Grade 1-2 hand-foot syndrome occurred in 4 patients. In conclusion, the combination of MMC and S-1 as third-line chemotherapy in patients with advanced CRC appears to be well tolerated but has poor activity.