Intracellular thiols like L-cysteine and glutathione play a critical role in the regulation of cellular processes. Escherichia coli has multiple L-cysteine transporters, which export L-cysteine from the cytoplasm into the periplasm. However, the role of L-cysteine in the periplasm remains unknown. Here we show that an L-cysteine transporter, YdeD, is required for the tolerance of E. coli cells to hydrogen peroxide. We also present evidence that L-cystine, a product from the oxidation of L-cysteine by hydrogen peroxide, is imported back into the cytoplasm in a manner dependent on FliY, the periplasmic L-cystine-binding protein. Remarkably, this protein, which is involved in the recycling of the oxidized L-cysteine, is also found to be important for the hydrogen peroxide resistance of this organism. Furthermore, our analysis of the transcription of relevant genes revealed that the transcription of genes encoding FliY and YdeD is highly induced by hydrogen peroxide rather than by L-cysteine. These findings led us to propose that the inducible L-cysteine/L-cystine shuttle system plays an important role in oxidative stress tolerance through providing a reducing equivalent to the periplasm in E. coli.