Oxidative stress is associated with the pathophysiology of many degenerative human diseases, including Alzheimer's disease, atherosclerosis, Parkinson's disease, and cancers. We discovered in our previous study that thioproline (SPro), a proline analogue, is generated in oxidant-exposed cells. With the prior observation that SPro served as an efficient nitrile trapping agent, we tested in this study the hypothesis that this oxidative stress generated cysteine-formaldehyde adduct, SPro, may serve as an antioxidant protecting cells from oxidative stress. Interestingly, results showed that HeLa cells cultured in SPro-supplemented culture media are more tolerant of oxidative stress, indicated by a dosage-dependent increase in cell viability. Investigation of the molecular mechanism of the observed increase in cell tolerance to oxidative stress revealed SPro acting as an effective antioxidant by sacrificial oxidation. Results also showed that SPro had been incorporated into cellular proteins and induced changes in protein expression profiles of treated cells. Despite being yet to determine the participation of individual factors to the observed increase of cell tolerance to oxidative stress, this study sheds light on the potential use of SPro as a dietary supplement for protecting humans from oxidative stress-associated degenerative human diseases.