Diclofenac is one of the most widely prescribed nonsteroidal anti-inflammatory drugs worldwide. It is frequently detected in surface waters; however, whether this pharmaceutical poses a risk to aquatic organisms is debated. Here we quantified the uptake of diclofenac by the fathead minnow (Pimephales promelas) following aqueous exposure (0.2-25.0 μg L-1) for 21 days, and evaluated the tissue and biomolecular responses in the kidney. Diclofenac accumulated in a concentration- and time-dependent manner in the plasma of exposed fish. The highest plasma concentration observed (for fish exposed to 25 μg L-1 diclofenac) was within the therapeutic range for humans. There was a strong positive correlation between exposure concentration and the number of developing nephrons observed in the posterior kidney. Diclofenac was not found to modulate the expression of genes in the kidney associated with its primary mode of action in mammals (prostaglandin-endoperoxide synthases) but modulated genes associated with kidney repair and regeneration. There were no significant adverse effects following 21 days exposure to concentrations typical of surface waters. The combination of diclofenac's uptake potential, effects on kidney nephrons and relatively small safety margin for some surface waters may warrant a longer term chronic health effects analysis for diclofenac in fish.