The improper maintenance of the bioactivated form of vitamin-D (1α,25(OH)2D) may result in vitamin-D insufficiency and therefore compromise the absorption of dietary calcium. A significant regulator of vitamin-D metabolism is the inactivating function of the mitochondrial enzyme cytochrome P450 24A1 (CYP24A1). In humans, CYP24A1 carries out hydroxylation of carbon-23 (C23) or carbon-24 (C24) of the 1α,25(OH)2D side chain, eventually resulting in production of either an antagonist of the vitamin-D receptor (C23 pathway) or calcitroic acid (C24 pathway). Despite its importance to human health, the human isoform (hCYP24A1) remains largely uncharacterized due in part to the difficulty in producing the enzyme using recombinant means. In this study, we utilize a cleavable fusion with the cognate redox partner, human Adx (hAdx), to stabilize hCYP24A1 during production. The subsequent cleavage and isolation of active hCYP24A1 allowed for an investigation of substrate and analog binding, enzymatic activity, and redox partner recognition. We demonstrate involvement of a nonpolar contact involving Leu-80 of hAdx and a nonconserved proximal surface of hCYP24A1. Interestingly, shortening the length of this residue (L80V) results in enhanced binding between the CYP-Adx complex and 1α,25(OH)2D yet unexpectedly results in decreased catalysis. The same mutation has a negligible effect on rat CYP24A1 (a C24-hydroxylase), indicating the presence of a species-specific requirement that may correlate with differences in regioselectivity of the reaction. Taken together, this work presents an example of production of a challenging human CYP as well as providing details regarding hydrophobic modulation of a CYP-Adx complex that is critical to human vitamin-D metabolism.