Within the family of NADPH oxidases, Nox4 is unique as it is predominantly localized in the endoplasmic reticulum, has constitutive activity and generates H2O2. We hypothesize that these features are consequences of a so far unidentified Nox4-interacting protein. Blue native gel electrophoresis of solubilized membranes from HEK293 cells (overexpressing Nox4) separated macromolecular complexes containing Nox4. The combination of native gel electrophoresis and quantitative mass spectrometry was applied to identify proteins that co-migrate with Nox4. In addition, the data set contains information about macromolecular protein complexes in human cellular membranes that are stable to the solubilization with digitonin.