Bifenthrin, a pyrethroid insecticide, undergoes oxidative metabolism leading to the formation of 4′-hydroxy-bifenthrin (4′-OH-BIF) and hydrolysis leading to the formation of TFP acid in rat and human hepatic microsomes. In this study, age-dependent metabolism of bifenthrin in rats and humans were determined via the rates of formation of 4′-OH-BIF and TFP acid following incubation of bifenthrin in juvenile and adult rat (PND 15 and PND 90) and human (< 5 years and > 18 years) liver microsomes. Furthermore, in vitro hepatic intrinsic clearance (CLint) of bifenthrin was determined by substrate consumption method in a separate experiment. The mean Vmax(± SD) for the formation of 4′-OH-BIF in juvenile rat hepatic microsomes was 25.0 ± 1.5 pmol/min/mg which was significantly lower (p < 0.01) compared to that of adult rats (86.0 ± 17.7 pmol/min/mg). However, the mean Km values for juvenile (19.9 ± 6.6 μM) and adult (23.9 ± 0.4 μM) rat liver microsomes were similar. On the other hand, in juvenile human hepatic microsomes, Vmax for the formation of 4′-OH-BIF (73.9 ± 7.5 pmol/min/mg) was significantly higher (p < 0.05) than that of adults (21.6 ± 0.6 pmol/min/mg) albeit similar Km values (10.5 ± 2.8 μM and 8.9 ± 0.6 μM) between the two age groups. The trends in the formation kinetics of TFP acid were similar to those of 4′-OH-BIF between the species and age groups, although the differences between juveniles and adults were less pronounced. The data also show that metabolism of bifenthrin occurs primarily via oxidative pathway with relatively lesser contribution (˜ 30%) from hydrolytic pathway in both rat and human liver microsomes. The CLint values for bifenthrin, determined by monitoring the consumption of substrate, in juvenile and adult rat liver microsomes fortified with NADPH were 42.0 ± 7.2 and 166.7 ± 20.5 μl/min/mg, respectively, and the corresponding values for human liver microsomes were 76.0 ± 4.0 and 21.3 ± 1.2 μl/min/mg, respectively. The data suggest a major species difference in the age dependent metabolism of bifenthrin. In human liver microsomes, bifenthrin is metabolized at a much higher rate in juveniles than in adults, while the opposite appears to be true in rat liver microsomes.