P450 oxidoreductase (POR) has a pivotal role in facilitating electron transfer from NADPH to microsomal cytochrome P450 (CYP) enzymes, including the steroidogenic enzymes CYP17A1 and CYP21A2. Mutations in POR have recently been shown to cause congenital adrenal hyperplasia (CAH) with apparent combined CYP17A1 and CYP21A2 deficiency that comprises a variable clinical phenotype including glucocorticoid deficiency, ambiguous genitalia and craniofacial malformations. To dissect structure-function relationships potentially explaining this phenotypic diversity we investigated whether specific POR mutations have differential effects on CYP17A1 and CYP21A2. We compared the impact of missense mutations encoding for single amino acid changes in three distinct regions of the POR molecule: firstly, Y181D and H628P close to the central electron transfer area, secondly, S244C located within the hinge close to the FAD and FMN domains of POR, and thirdly, A287P that is clearly distant from the two other regions. Functional analysis employing a yeast microsomal assay with co-expression of human CYP17A1 or CYP21A2 with wild-type or mutant human POR revealed equivalent decreases in CYP17A1 and CYP21A2 activities by Y181D, H628P and S244C. By contrast, A287P had a differential inhibitory effect, with decreased catalytic efficiency (Vmax/Km) for CYP17A1 while CYP21A2 retained near normal activity. In vivo analysis of urinary steroid excretion by gas chromatography/mass spectrometry in 11 patients with POR mutations showed that A287P homozygous patients had the highest corticosterone/cortisol metabolite ratios, further indicative of preferential inhibition of CYP17A1. These findings provide novel mechanistic insights into the redox regulation of human steroidogenesis. Differential interaction of POR with electron-accepting CYP enzymes may explain the phenotypic variability in POR deficiency, with further implications for hepatic drug metabolism by POR-dependant CYP enzymes.