Background: A clinical trial of S-1 with leucovorin (S-1/LV) in metastatic colorectal cancer (CRC) patients demonstrated promising efficacy; however, the gastrointestinal toxicities were so severe that it has not been applied in the clinical setting. On the other hand, alternate-day administration of S-1 has been proposed to attenuate the adverse events without reducing its anticancer activity. Our present study was conducted to confirm the feasibility of alternate-day administration of S-1/LV in in vivo xenograft tumor models. Methods: Mice were treated with S-1/LV in a daily group (2 weeks of administration followed by 2 weeks of withdrawal) or an alternate-day group (administration on alternate days for 4 weeks), then the mice were killed and the xenograft tumors were resected. We compared body weight changes, condition of feces, mucosal injury and myelosuppression and assessed adverse reactions, tumor volume, tumor growth inhibition (TGI) and expression of Ki67, TUNEL, cIAP2 and XIAP to evaluate the antitumor activity and tumor apoptosis. Results: Severe weight loss, diarrhea, mucosal injury and myelosuppression were observed only in the daily group; however, some myelosuppression was also observed in the alternate-day group. The TGI in the alternate-day group was better than in the daily group, possibly resulting from apoptosis due to the suppression of cIAP2 but not XIAP. Conclusion: Our findings suggest that alternate-day administration of S-1/LV for CRC treatment can achieve high antitumor activity without severe adverse reactions, and we propose that clinical trials with this regimen should be conducted in CRC patients.