Tobacco smoke is a major contributing factor for chronic obstructive pulmonary disease (COPD) and exposure leads to oxidative stress, which contributes to alveolar wall destruction, mucus hyper secretion, inflammation and defective tissue repair. Ergothioneine (ET), an amino acid transported across cell membranes by OCTN1 (SLC22A4), has been shown to exhibit antioxidant capacities. The objective of this study was to investigate the role of OCTN1-transported ET in tobacco smoke-induced oxidative stress. For in vitro studies, human NCI-H441 cells were cultured for at least 5 days in the presence or absence ET and then exposed to cigarette smoke extract (CSE). Reactive oxygen species were measured using H2DCFDA assay. Enzymes involved in oxidative stress defence (e.g. catalase, thioredoxin, sulfiredoxin 1) and markers of xenobiotic defence (e.g. MRP1, BCRP and PXR) were studied by q-PCR and immunoblot. Moreover, Octn1 knock-out mice and wildtype controls were exposed for 3 and 6 months to second-hand cigarette smoke and changes in lung morphology were assessed in haematoxylin-eosin (HE) stained lung sections. In addition, total cell numbers and types in broncho-alveolar lavage fluid (BALF) were counted and inflammation markers quantified by q-PCR and ELISA. q-PCR and immunoblot revealed elevated expression levels of catalase, thioredoxin and sulfiredoxin 1 as well as PXR, MRP1 and BCRP following ET treatment of NCI-H441 cells. Moreover, lower levels of oxidative stress were observed in cells, which were cultured in the presence of ET prior to CSE exposure. When exposed to room air, knock-out mice showed little differences compared to wildtype animals. However, numbers of total cells and PNMs in BALF as well as increased alveolar damage and increased inflammatory markers were observed in knock-out animals compared to control mice, when exposed to second-hand smoke. These data suggest that ET can protect lung epithelial cells from oxidative damage and consequently, variants of OCTN1 might play a role in the pathogenesis of tobacco smoke-induced COPD by regulating ET transport.