This study was conducted to investigate the inhibitory effects of saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), and polyunsaturated fatty acids (PUFAs) on cytochrome P450 3A (CYP3A). The inhibition of 6β-hydroxy testosterone formation from testosterone in rat liver microsomes was used as an index of CYP3A activity. In the present study, among the three types of fatty acids, the rank order of inhibitory effects of fatty acids was SFAs<MUFAs<PUFAs. Among the four PUFAs examined (linoleic acid, γ-linolenic acid, retinoic acid, and docosahexaenoic acid; DHA), the highest inhibitory effect of CYP3A-catalyzed testosterone metabolism was observed with DHA, which inhibited testosterone metabolism by 94%. The percentage inhibition (I) of fatty acids on testosterone 6β-hydroxylation was predicted from the number of double bonds and the carbon chain length of the fatty acids. The correlation between the predicted and observed I values yielded the r² values of 0.759 and 0.907 using the linear and non-linear equations, respectively. Moreover, the goodness-of-fit assessed using the mean absolute error (MAE) was 17.6 with the linear model and 11.3 with the non-linear model. These observations suggested that fatty acids showed the variable inhibitory effects on CYP3A-mediated testosterone oxidation and that the number of double bonds, as well as the carbon chain lengths of fatty acids, may be important in the inhibition of CYP3A.