Toxicological effects of neonicotinoid insecticides (NNIs) have been reported for mammals, such as humans, rats, and mice. However, there are limited reports on their toxic effects on wild mammals. To predict NNI‐induced toxic effects on wild mammals, it is necessary to determine the exposure levels and metabolic ability of these species. We considered that raccoons could be an animal model for evaluating NNI‐induced toxicities on wildlife because they live near agricultural fields and eat crops treated with NNIs. The objective of the present study was to estimate the effects of NNI exposure on wild raccoons. Urinary concentrations of NNI compounds (n = 59) and cytochrome P450‐dependent metabolism of NNIs (n = 3) were evaluated in wild raccoons captured in Hokkaido, Japan, in 2020. We detected either one of the six NNIs or one metabolite, including acetamiprid, imidacloprid, clothianidin, dinotefuran, thiacloprid, thiamethoxam, and desmethyl‐acetamiprid in 90% of raccoons (53/59); the average cumulative concentration of the seven NNI compounds was 3.1 ng/ml. The urinary concentrations were not much different from those reported previously for humans. Furthermore, we performed an in vitro assessment of the ability of raccoons to metabolize NNIs using hepatic microsomes. The amounts of NNI metabolites were measured using liquid chromatography–electrospray ionization–tandem mass spectrometry and compared with those in rats. Raccoons showed much lower metabolic ability; the maximum velocity/Michaelis–Menten constant (Vmax/Km) values for raccoons were one‐tenth to one‐third of those for rats. For the first time, we show that wild raccoons could be frequently exposed to NNIs in the environment, and that the cytochrome P450‐dependent metabolism of NNIs in the livers of raccoons might be low. Our results contribute to a better understanding of the effects of NNIs on raccoons, leading to better conservation efforts for wild mammals. Environ Toxicol Chem 2022;41:1865–1874. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.