Maraviroc is an anti‐HIV drug that acts by blocking viral entry into target cells. Although maraviroc is known to be metabolized by the cytochromes P450 (CYP) to several products, a comprehensive analysis of the enzymes involved has yet to be reported; therefore, this work sought to identify the CYPs involved in the oxidative metabolism of maraviroc. Using a panel of cDNA‐expressed CYPs it was determined that the major oxidative metabolite of maraviroc (detected using liquid chromatography‐mass spectrometry) was primarily formed by CYP3A5. Tandem mass spectrometry analysis indicated that the CYP3A5‐mediated oxygen insertion occurred on the difluorocyclohexane ring of maraviroc. For this reaction, the Vmax for CYP3A4 and CYP3A5 was 0.04 and 0.93 pmole/min/pmole P450, respectively. In addition, the Km values were 11.1 μM and 48.9 μM, respectively. Human liver microsomes isolated from donors homozygous for the CYP3A5*3 allele, which results in low expression of CYP3A5, exhibited a 79% decrease in formation of this metabolite as compared to production by human liver microsomes isolated from donors homozygous for the CYP3A5*1 allele. In conclusion, the major oxidative metabolite of maraviroc was found to be formed primarily by CYP3A5. These results indicate that maraviroc may be used as a tool to distinguish the activity of CYP3A5 from that of CYP3A4. Support: PhRMA Foundation Research Starter Grant to N.B.