Cytochrome P450 reductase (CPR) is a tethered membrane protein which transfers electrons from NADPH to microsomal P450s. We show that the lipid bilayer has a role in defining the redox potential of the CPR flavin domains. In order to quantitate the electrochemical behavior of this central redox protein, full-length CPR was incorporated into soluble nanometer scale discoidal membrane bilayers (nanodiscs), and potentials were measured using spectropotentiometry. The redox potentials of both FMN and FAD were found to shift to more positive values when in a membrane bilayer as compared to a solubilized version of the reductase. The potentials of the semiquinone/hydroquinone couple of both FMN and FAD are altered to a larger extent than the oxidized/semiquinone couple which is understood by a simple electrostatic model. When anionic lipids were used to change the membrane composition of the CPR-nanodisc, the redox potential of both flavins became more negative, favoring electron transfer from CPR to cytochrome P450.