Abstract: Cytochrome P450s （CYPs） is the most important enzyme for drug metabolism in animals. Inappropriate coadministration often causes treatment failure in fisheries owing to induction of CYP activity. However, limited information is available about CYP induction in fish. In order to supply useful information on CYP induction, the present study assessed the effects of fish specific CYP inducers on difloxacin （DIF） metabolism and the enzyme kinetics in kidney cell of grass carp （Ctenopharyngodon idellus） （CIK） by reversed-phase high-performance liquid chromatography （RP-HPLC）. The enzymatic equations of control and groups induced by β-naphthoflavone （BNF）were 1/V= 0.137 5 ×1/［S］+0.003 and 1/V=0.024 5×1/［S］+0.001 3. Results demonstrated that the amounts of DIF metabolism were increased by 1 fold and enzymatic parameters Clint and V_max of DIF metabolism were significantly increased by 7 and 2 folds due to BNF pretreatment. BNF is the specific inducer of CYP1A in fish. Therefore, CYP1A may be responsible for DIF metabolism. This study provides instructive information to ensure treatment success in fisheries medication with two or more drugs.