Neonicotinoid pesticides (NNs) act as agonists on nicotinic acetylcholine receptors (nAChRs) of insects, and there have been concerns about the effects of NNs on the health of mammals. Since nAChRs are expressed in immune cells, it is possible that NNs disturb the immune system. However, few reports have examined the immunotoxicity of clothianidin (CLO), a widely-used NN. Here, we report the effects of CLO on immune organs and type IV allergic reactions in ear auricles. We orally administered CLO at 0, 30 and 300 mg/kg/day (CLO-0, 30 and 300) to Sprague-Dawley rats for 28 days. The effects were evaluated by organ and body weights, histopathology, and immunohistochemistry (TCRαβ, CD4, CD8, CD11b, CD68, CD103). In addition, some cecal contents were subjected to preliminary gut microbiota analysis, because microbiota contribute to host homeostasis, including the immunity. Our results showed loose stool, suppression of body weight gain, significant changes in organ weights (thymus: decreased; liver: increased) and changes of the gut microbiota in the CLO-300 group. There were no obvious histopathological changes in immune organs. Granulomas of the ear auricles were found in one rat of each of the CLO-30 and 300 groups, but CLO had no apparent effect on the thickness or immunohistochemistry in the ear auricles. We present new evidence that CLO affects the thymus and intestine, and might enhance the local inflammatory response. These findings should contribute to the appropriate evaluation of the safety of NNs in the future.