S-1 and irinotecan combination is attractive for breast cancer refractory to anthracyclines and taxanes. Patients with advanced human epidermal growth factor receptor 2 (HER2)-negative breast cancer previously treated with anthracyclines and taxanes were eligible. Patients with brain metastases and homozygous for UGT1A1 *6 or *28 or compound heterozygous (*6/*28) were excluded. A dose-escalation design was chosen for the phase I portion (level 1: irinotecan 80mg/m(2)days 1-8 and S-1 80mg/m(2)days 1-14 every 3weeks; level 2: irinotecan 100mg/m(2) and S-1 80mg/m(2)). Study objectives included determination of the recommended dose for phase II, response rate, progression-free survival (PFS), and safety. Pharmacokinetics and CD34(+) circulating endothelial cells (CECs) as pharmacodynamics were also analyzed. Thirty-seven patients were included. One patient at each level developed dose-limiting toxicities; therefore, level 2 was the recommended dose for phase II. Diarrhea was more common in patients possessing a *6 or *28 allele compared with wild-type homozygous patients (46% and 25%). Among 29 patients treated at level 2, PFS was longer for UGT1A1 wt/*6 and wt/*28 patients than for wt/wt patients (12 vs. 8months, P=0.06). PFS was significantly longer in patients with a larger-than-median SN-38 area under the curve (AUC) than in those with a smaller AUC (P=0.039). There was an association between clinical benefit and reduction in baseline CD34(+) CECs by S-1 (P=0.047). The combination of irinotecan and S-1 is effective and warrants further investigation.