There is concern regarding oil sands process water (OSPW) produced by the oil sands industry in Alberta, Canada. Little is known about the potential for OSPW, and naphthenic acids (NAs), which are the primary persistent and toxic constituents of OSPW, to affect endocrine systems. Although ozonation significantly reduces concentrations of NAs and OSPW toxicity, it was hypothesized that oxidation of OSPW might produce hydroxylated products with steroidogenic activity. Therefore, untreated and ozone treated OSPW were examined for effects on sex steroid production using the H295R Steroidogenesis Assay. Untreated OSPW significantly decreased testosterone (T) and increased 17β-estradiol (E2) concentrations at OSPW dilutions greater or equal to 10-fold. This effect was mainly due to decreased E2 metabolism. Analysis of CYP19A (aromatase) mRNA abundance and enzyme activity suggested that induction of this enzyme activity may have also contributed to these effects. Reduction of parent NA concentrations by 24% or 85% decreased the effect of OSPW on E2 production. Although T production remained significantly reduced in cells exposed to ozone treated OSPW, the effect was diminished. Aromatase mRNA abundance and enzyme activity were significantly greater in cells exposed to ozone treated OSPW, however the magnitude was less than in cells exposed to untreated OSPW. No change of E2 metabolism was observed in cells exposed to ozone treated OSPW, which may account for recovery of E2 levels. The results indicate that OSPW exposure can decrease E2 and T production, but ozonation is an effective treatment to reduce NA concentrations in OSPW without increasing affects on steroidogenesis.