Under oxidative stress, cytoprotective genes are initiated and regulated by various transcription factors in order to minimize oxidative damage to the cell. Nuclear factor erythroid-2-like 1 (Nrf1) is a transcription factor, a vital regulators of antioxidant and detoxification genes through the antioxidant response element (ARE). This study aimed to determine whether Nrf1 is regulated, in an oxygen-dependent manner, by hydroxylation. A potential hydroxylation site was identified on Nrf1 that may be targeted by the Prolyl Hydroxylase 2 (PHD2). Oxygen-dependent hydroxylation of Nrf1 may affect it function under low oxygen (hypoxic (1% O2)) conditions when hydroxylation is absent. The potential hydroxylation site was mutated in Nrf1 and wildtype and mutant proteins were overexpressed in human embryonic kidney (HEK293T) cells. We hypothesized that Nrf1 is hydroxylated by PHD2 in an oxygen-dependent manner and this affects Nrf1 function and protein stability.