Imidacloprid is a widely used systemic neonicotinoid insecticide. The aim of this study is to identify potential harmful effects of imidacloprid in freshwater mussels (Unio mancus) which were exposed to sub-lethal concentrations (1, 10, 100, and 1000 µg AI L-1) for 96 h. For this aim, glutathione s-transferase (GST) activity, carboxylesterase (CaE) activity, acetylcholinesterase (AChE) activity, glutathione reductase (GR) activity, reduced glutathione (GSH) level, and malondialdehyde (MDA) level were evaluated as biochemical markers of exposure in the gills and digestive glands. The actual imidacloprid concentrations in the test waters were determined by LC-MS/MS analysis. The results showed that the AChE, GR, and CaE activities in the digestive glands significantly decreased at different exposure concentrations, while the MDA level significantly increased at the highest exposure concentration (p<0.05). Considering the results in the gills, the lowest exposure concentration caused inhibition in AChE activity, while the highest exposure concentration caused significant induction in GST activity, GSH level and MDA level (p<0.05). In addition, the measured imidacloprid concentrations were determined to be about 80% of the nominal imidacloprid concentrations. The data obtained from this study indicated that acute imidacloprid exposure caused biochemical alterations related to oxidative stress in mussels.