The chemical reactivity of acyl glucuronide (AG) has been thought to be associated with the toxic properties of drugs containing carboxylic acid moieties, but there has been no direct evidence that AG formation was related to the toxicity. In the present study, the cytotoxicity and genotoxicity of AGs were investigated. Human embryonic kidney (HEK) 293 cells stably expressing UDP-glucuronosyltransferase (UGT) 1A3 (HEK/UGT1A3) were constructed to assess the cytotoxicity of AGs, and HEK/UGT1A4 cells were also used as a negative reference. After exposure to nonsteroidal anti-inflammatory drugs (NSAIDs) such as naproxen (1 mM), diclofenac (0.1 mM), ketoprofen (1 mM), or ibuprofen (1 mM) for 24 h, HEK/UGT1A3 cells produced AG in a time-dependent manner. However, HEK/UGT1A4 cells hardly produced AG. The cytotoxicity of HEK/UGT1A3 cells was not increased compared with that of HEK/UGT1A4 cells. In addition, the AG formed in NSAID-treated human hepatocytes was decreased from one-third to one-ninth by treatment with (-)-borneol, an inhibitor of acyl glucuronidation, but the cytotoxicity was increased. These results indicated that AG formation reflected the detoxification process in human hepatocytes. Furthermore, the possibility of genotoxicity from the AG formed in NSAID-treated HEK/UGT cells was investigated by the comet assay, and DNA damage was not detected in any HEK/UGT cell lines. In conclusion, the in vitro cytotoxic and genotoxic effects of the AGs of NSAIDs were investigated and AG was not found to be a causal factor in the toxicity.