The glucose-regulated stress response in mammalian cells is characterized by the increased synthesis of glucose-regulated proteins (GRPs). In this study, we found that GRP-inducing conditions in culture led to induction of resistance to the topoisomerase I-targeted drug camptothecin in human colon cancer HT-29 and ovarian cancer A2780 cells. The induction of camptothecin resistance was accompanied by decreased levels of camptothecin-induced cleavable complexes, as measured by a topoisomerase I band depletion assay. However, topoisomerase I protein levels were the same in both stressed and non-stressed cells. Furthermore, when isolated nuclei from stressed and non-stressed cells were treated with camptothecin, similar levels of cleavable complexes were obtained, suggesting that the activity of topoisomerase I did not change in stressed cells. In contrast, intracellular accumulation of camptothecin decreased in stressed cells. Our results indicate that stress-induced camptothecin resistance could be explained by reduced camptothecin accumulation, leading to decreased numbers of cleavable complexes, without quantitative or qualitative changes in topoisomerase I levels. In addition, cell cycle analysis revealed that the GRP-inducing treatments resulted in an accumulation of G1/G0-phase cells. As camptothecin shows an S-phase-specific cytotoxicity, the G1/G0-phase accumulation is another mechanism for camptothecin resistance. Since a glucose-regulated response is produced by hypoxia and nutrient deprivation that occur naturally in solid tumors, the resistance observed here can occur in some solid tumors and can be an obstacle to chemotherapy.