The nonsteroidal anti-inflammatory drug (NSAID) sulindac and the ornithine decarboxylase (ODC) antagonist difluoromethylornithine (DFMO), individually and together, are effective inhibitors of colon carcinogenesis. However, chronic use of sulindac is associated with significant side effects. We evaluated the chemopreventive efficacy of phospho-sulindac (P-S, OXT-328), an apparently safe derivative of sulindac, together with DFMO, in HT-29 human colon cancer xenografts. Nude mice were divided into four groups as follows: group 1 received vehicle (corn oil); group 2 received P-S (100 mg/kg/d) by oral gavage; group 3 received DFMO (2% in drinking water); and group 4 received P-S (100 mg/kg/d) by gavage plus DFMO (2% in drinking water; P-S/DFMO). Eighteen days after implantation, compared with controls, tumor volume was inhibited 65.9% by P-S, 52.9% by DFMO, and 70.9% by P-S/DFMO (P < 0.01 for all). P-S/DFMO reduced cell proliferation 27.1% and increased apoptosis 38.9% compared with controls (P < 0.05 for both). Compared with controls, P-S reduced the levels of thioredoxin-1 (Trx-1) and thioredoxin reductase (TrxR), whereas DFMO reduced polyamine content (putrescine and spermidine) and TrxR levels. Importantly, P-S/DFMO decreased putrescine and spermidine levels and the expression of Trx-1, TrxR, and cyclooxygenase (COX) 2. Of these molecular targets, TrxR most consistently correlated with tumor growth. Study results show that P-S/DFMO is an efficacious drug combination for colon cancer prevention and also show the safety of P-S, which may overcome the limiting side effects of conventional sulindac. P-S/DFMO has an intricate mechanism of action extending beyond polyamines and including the thioredoxin system, an emerging regulator of chemoprevention. P-S/DFMO merits further evaluation.