The compound 3,4-dichlorophenyl-propenoyl-sec.-butylamine (3,4-DCPB) is an antiepileptic drug. The purpose of the present research was to identify cytochrome P450 (CYP450) responsible for the metabolism of 3,4-DCPB and evaluate the effects of 3,4-DCPB on the activities of CYP450 enzymes. 3,4-DCPB was incubated with rat liver microsomes (RLMs) plus six CYP450 enzyme-specific inhibitors, or six recombinant human CYP450 enzymes (rhCYP450s). The concentrations of 3,4-DCPB and six CYP450 enzyme-activities probe drugs were detected by high-performance liquid chromatographic (HPLC). The results showed that the prototype of 3,4-DCPB was metabolized by multiple CYP450 enzymes into three metabolites, and the predominant isoforms were CYP2D6 (metabolite M1), CYP1A2 (M2), CYP2C19 and CYP3A4 (M3), respectively., in the presence of β-NADPH (1 mM) in RLMs or rhCYP450s. Compared with the control (PB−), phenobarbital pre-treatment (PB+) significantly enhanced levels (all of p < 0.01) of hydroxylmethytobutamide (CYP2C9), 4-hydroxy-mephenytoin (CYP2C19), acetaminophen (CYP1A2), 6-hydroxychlorzoxazone (CYP2E1) and oxidized nifedipine (CYP3A4), respectively, in spite of dextrophan (CYP2D6) was not markedly enhanced in RLMs. Conversely, the inhibitory ratios of 3,4-DCPB (16 μg/mL, 59 μM) on the activities of CYP2C9, CYP2C19, CYP1A2 and CYP2D6 were 97.6%, 59.0%, 53.5% and 36.5%, respectively. However, CYP2E1 (both of PB− and PB+) and CYP3A4 (PB+) were not inhibited by 3,4-DCPB in RLMs. In conclusion, the present study showed that 3,4-DCPB was metabolized by multiple CYP450 enzymes. 3,4-DCPB inhibited the activities of CYP2C9, CYP2C19, CYP1A2 and CYP2D6, rather than that CYP2E1 and CYP3A4 enzymes, suggesting that the different effects of 3,4-DCPB on the CYP450 enzymes might influence metabolic drug-drug interaction in antiepileptics therapy.