2-Deoxy-d-ribose (dRib) is known to induce oxidative damage and apoptosis by depleting GSH content in several types of cells. However, little is known about how dRib depletes intracellular GSH content. We hypothesized that dRib would deplete GSH content through inhibition of cystine uptake via system χc-. HIT-T15 cells were incubated with various concentrations of dRib, 2-mercaptoethanol (2-ME), Sulfasalazine (SASP), and homocysteic acid (HCA). We measured intracellular uptake of l-[14C]cystine, GSH content, and cell viability. Stimulation with dRib dose- and time-dependently decreases intracellular l-[14C]cystine uptake and GSH content. The addition of 2-ME, a cystine uptake enhancer, fully recovered the dRib-induced decreases in l-[14C]cystine uptake, GSH content, and cell viability. The 2-ME promoted the l-[14C]cystine uptake even in the absence of extracellular Na+, probably suggesting the involvement of the system χc-. SASP and HCA, system χc- inhibitors, significantly reduced l-[14C]cystine uptake and GSH content like dRib did, and 2-ME also restored the reductions. These findings suggest that dRib depletes intracellular GSH content through inhibition of cystine transport, and this inhibition may be mediated by system χc-. Additional experiments using a gene overexpression technique is necessary to further clarify the mechanism.