Per- and polyfluoroalkyl substances (PFAS) are ubiquitous environmental pollutants that are highly stable synthetic organofluorine compounds. One congener perfluorooctanoic acid (PFOA) can be detected in nearly all humans and is recognized as an endocrine disrupting chemical (EDC). EDCs disrupt hormone synthesis and metabolism and receptor function. One mechanism of steroid hormone action is the pre-receptor regulation of ligand access to steroid hormone receptors by aldo-keto reductases. Here we report PFOA inhibition of AKR family 1 member C2 (AKR1C2), leading to dysregulation of androgen action. Spectrofluorimetric inhibitor screens identified PFOA as a competitive and tight binding inhibitor of AKR1C2, whose role is to inactivate 5α-dihydrotestosterone (5α-DHT). Further site directed mutagenesis studies along with molecular docking simulations revealed the importance of residue Valine 54 in mediating AKR1C2 inhibitor specificity. Binding site restrictions were explored by testing inhibition of other related PFAS chemicals, confirming that steric hinderance is a key factor. Furthermore, radiochromatography using HPLC and in line radiometric detection confirmed the accumulation of 5α-DHT as a result of PFOA inhibition of AKR1C2. We showed that PFOA could enhance the transactivation of AR in reporter genes assays in which 5α-DHT metabolism was blocked by AKR1C2 inhibition in HeLa cells. Taken together, these data suggest PFOA has a role in disrupting androgen action through inhibiting AKR1C2. Our work identifies an EDC function for PFOA not previously revealed.