Thymidine phosphorylase (TP), a rate-limiting enzyme in thymidine catabolism, plays a pivotal role in tumor progression; however, the mechanisms underlying this role are not fully understood. Here, we found that TP-mediated thymidine catabolism could supply the carbon source in the glycolytic pathway and thus contribute to cell survival under conditions of nutrient deprivation. In TP-expressing cells, thymidine was converted to metabolites, including glucose 6-phosphate, lactate, 5-phospho-α-D-ribose 1-diphosphate, and serine, via the glycolytic pathway both in vitro and in vivo. These thymidine-derived metabolites were required for the survival of cells under low-glucose conditions. Furthermore, activation of thymidine catabolism was observed in human gastric cancer. These findings demonstrate that thymidine can serve as a glycolytic pathway substrate in human cancer cells