The effects of hydraulic fracturing (HF) flowback and produced water (HF-FPW), a complex saline mixture of injected HF fluids and deep formation water that return to the surface, was examined in rainbow trout (Oncorhynchus mykiss). Exposure to HF-FPWs resulted in significant induction of ethoxyresorufin-O-deethylase (EROD) activity in both liver and gill tissues. Increased lipid peroxidation via oxidative stress was also detected by thiobarbituric acid reactive substances (TBARS) assay. The mRNA expressions of a battery of genes related to biotransformation, oxidative stress, and endocrine disruption were also measured using quantitative real-time polymerase chain reaction (Q-RT-PCR). The increased expression of cyp1a (2.49 ± 0.28-fold), udpgt (2.01 ± 0.31-fold), sod (1.67 ± 0.09-fold), and gpx (1.58 ± 0.10-fold) in raw sample exposure group (7.5%) indicated elevated metabolic enzyme activity, likely through the aryl hydrocarbon receptor pathway, and generation of reactive oxygen species. In addition, the elevated vtg and era2 expression demonstrated endocrine disrupting potential exerted by HF-FPW in rainbow trout. The overall results suggested HF-FPW could cause significant adverse effects on fish, and the organic contents might play the major role in its toxicity. Future studies are needed to help fully determine the toxic mechanism(s) of HF-FPW on freshwater fish, and aid in establishing monitoring, treatment, and remediation protocols for HF-FPW.