In vivo, mammalian cells reside in an environment of 0.5-10% O2 (depending on the tissue location within the body), whilst standard in vitro cell culture is carried out under room air. Little is known about the effects of this hyperoxic environment on treatment-induced oxidative stress, relative to a physiological oxygen environment. In the present study we investigated the effects of long-term culture under hyperoxia (air) on photodynamic treatment. Upon photodynamic irradiation, cells which had been cultured long-term under hyperoxia generated higher concentrations of mitochondrial reactive oxygen species, compared with cells in a physioxic (2% O2) environment. However, there was no significant difference in viability between hyperoxic and physioxic cells. The expression of genes encoding key redox homeostasis proteins and the activity of key antioxidant enzymes was significantly higher after the long-term culture of hyperoxic cells compared with physioxic cells. The induction of antioxidant genes and increased antioxidant enzyme activity appear to contribute to the development of a phenotype that is resistant to oxidative stress-induced cellular damage and death when using standard cell culture conditions. The results from experiments using selective inhibitors suggested that the thioredoxin antioxidant system contributes to this phenotype. To avoid artefactual results, in vitro cellular responses should be studied in mammalian cells that have been cultured under physioxia. This investigation provides new insights into the effects of physioxic cell culture on a model of a clinically relevant photodynamic treatment and the associated cellular pathways.