Abstract: To investigate the change in CYP3A4 activity and its significance in hepatocarcinogenesis induced by aflatoxin B1 ( AFB1 ) in rats. Methods: Four-week old male Wistar rats were randomly divided into AFB1 and control groups. Rats in the AFB1 groups were intraperitoneally injected with AFB1 and dimethyl sulfoxide. During hepatocarcinogenesis, liver biopsies were performed on all animals on the 13th, 23rd, 33rd, 43rd, 53rd, and 63rd week of the experiment. The animals were sacrificed on the 73rd week and liver tissues were collected. Using the hepatic microsomal mixed-function oxidase enzyme system, the CYP3A4 activity was dynamically examined in liver samples via quantitative fluorescence spectrophotometry. Results: The hepatocellular carcinoma incidence in the AFB1 group ( 58.8%, 10/17 ) was significantly higher than in the control group ( 0.0%, 0/16 ), with P = 0.001. Both groups exhibited different levels of changes in CYP3A4 activity. The CYP3A4 activity in both groups  gradually increased from the 13th week of the experiment, with a peak value on the 23rd week. The activity then gradually decreased, and again increased on the 43rd week, forming double peaks. The CYP3A4 activities in the AFB1 group significantly decreased during the 13rd and 53rd weeks compared with those of the control group ( P = 0.000 ). The level of CYP3A4 activity in the AFB1 group was close but not statistically significant to that of the control group on the 63rd week ( P = 0.5086 ). Conclusion: The CYP3A4 activity was inhibited in rats during hepatocarcinogenesis. This inhibition may be due to the decreased carcinogen activation in cells and the change in protein expression caused by genetic polymorphisms.