Objective To study the effect of cefetamet hydrochloride injection on the activity of 3 kinds of cytochrome P450 (CYP450) isoforms (CYP1A2, CYP3A4 and CYP2E1) in rat liver microsomes. Methods The SD rats were randomly divided into two groups: control group and cefetamet hydrochloride (CH) group, with each group containing 3 male rats and 3 female rats. The CH group was injected with cefetamet hydrochloride into the tail vein at 50 mg/(kg·d), twice a day for 7 days. A HPLC method was used for simultaneous determination of the production of metabolites and the degradation of the prototype probe substrates of 3 kinds of CYP450 isoforms, so as to evaluate the activity of hepatic CYP450. The analytical column was Diamonsil C₁₈ column (150 mm×4.6 mm, 5 μm), with the flow rate being 1.0 mL/min. The mobile phase consisted of methanol (0.1% formic acid) (A)-water (0.1% formic acid)(B), 0-5 min: 18%A, 5-10 min: 18%-60%A, 10-15 min: 60%A and detected at 247 nm for determination of CYP1A2 activities; methanol (A)-water (0.02% formic acid)(B), 0-11 min: 40%-60%A and detected at 223 nm for determination of CYP3A4 activities; and methanol (A)-water, 0-10 min: 37%-75%A and detected at 287 nm for determination of CYP2E1 activities. Results Probe substrates and their metabolites showed good linearity within the determining range (r≥0.999 7). The precision of the method was <6% (n=5) and extraction recoveries were 83.2%-97.5%. After 7-day injection of CH,CYP3A4 activities were significantly different between the two groups (P<0.05); CYP1A2 and CYP2E1 activities were not significantly different between the two groups (P>0.05). Conclusion CH injection can significantly induce hepatic microsome CYP3A4 expression in SD rats, but has no induction or inhibition effect on CYP1A2 and CYP2E1, indicating that potential drug-drug interaction might occur when CH injection is co-administered with drugs metabolized by CYP3A4.