Cysteine levels are carefully regulated in mammals to balance metabolic needs against the potential for cytotoxicity. It has been postulated that one of the major regulators of intracellular cysteine levels in mammals is cysteine dioxygenase (CDO). Hepatic expression of this catabolic enzyme increases dramatically in response to increased cysteine availability and may therefore be part of a homeostatic response to shunt excess toxic cysteine to more benign metabolites such as sulfate or taurine. Direct experimental evidence, however, is lacking to support the hypothesis that CDO is capable of altering steady-state intracellular cysteine levels. In this study, we expressed either the wild-type (WT) or a catalytically inactivated mutant (H86A) isoform of CDO into HepG2/C3A cells (which do not express endogenous CDO protein) and cultured them in different concentrations of extracellular cysteine. WT CDO, but not H86A CDO, was capable of reducing intracellular cysteine levels in cells incubated in physiologically relevant concentrations of cysteine. WT CDO also decreased the glutathione pool and potentiated the toxicity of CdCl2. These results demonstrate that CDO is capable of altering intracellular cysteine levels as well as glutathione levels.