Neonicotinoids have been often detected in aquatic environment with high concentrations; however, little is known about their risk and fate to/in fish. This study systematically investigated the bio-uptake, tissue distribution and metabolism of neonicotinoids in zebrafish, taking clothianidin (CLO) as an example. The results revealed the uptake and elimination kinetics of CLO in whole fish and different tissues was very similar, and its bioconcentration factor (<1) indicates the low bioaccumulation potential in zebrafish. The highest accumulative tissues for CLO were found to be intestine and liver. Eight biotransformation products were identified in intestine and liver, and the metabolic pathways were found to be N-demethylation and nitro-reduction. The metabolic kinetics of two products (desmethyl clothianidin and clothianidin urea) revealed the metabolism of CLO mainly occurred in liver and intestine. This suggested that the hepatobiliary system played an important role in the metabolism and elimination of CLO. This study provides a comprehensive evaluation of the toxicokinetics of CLO in zebrafish, and these results can contribute to its ecological risk assessment.