Purpose: 4′-Thio-β-D-arabinofuranosylcytosine (4′-thio-ara-C), which has shown significant cytotoxicity against a panel of human tumor lines, was evaluated for antitumor activity against a spectrum of human tumor systems in mice. Methods: Antitumor activity was evaluated in 15 subcutaneously implanted human tumor xenografts. 4′-Thio-ara-C was administered intraperitoneally using either q1dx9 (daily treatment for nine consecutive days) or q4hx3/q1dx9 (three treatments each day separated by 4-h intervals for nine consecutive days). Results: 4′-Thio-ara-C exhibited an excellent spectrum of activity. Treatment with the compound was curative against HCT-116 colon, SW-620 colon, NCI-H23 NSCL, and CAKI-1 renal tumors and resulted in partial/complete regressions in the DLD-1 colon, NCI-H522 NSCL, DU-145 prostate, and PANC-1 pancreatic tumor models. Tumor stasis was noted for HT29 colon and NCI-H460 NSCL tumors. Tumor inhibition was observed for A549 NSCL, PC-3 prostate, LNCAP prostate, and MDA-MB-435 breast tumors. Of the 15 tumors examined, only CFPAC-1 pancreatic was unresponsive to the compound. In contrast, 1-β-D-arabinofuranosylcytosine was minimally active at best against CAKI-1 renal, HCT-116 Colon, NCI-H460 NSCL, and SW-620 colon tumors. Schedule- and route-dependency studies were conducted using the NCI-H460 NSCL tumor. The activity of 4′-thio-ara-C was independent of schedule when comparing q2dx5 (every other day for five treatments), q1dx9, and q4hx3/q1dx9 treatment schedules. 4′-Thio-ara-C was equally effective by the intravenous and intraperitoneal routes of administration, with the oral route being less efficacious. Conclusions: On the basis of these results, 4′-thio-ara-C appears to have a profile distinct from other nucleoside antitumor agents and is being advanced to clinical trials.