Background Tuberculosis (TB) is the leading opportunistic infection of people living with human immunodeficiency virus (HIV; PLWH). Cytochrome P450 (CYP) 2B6 and ATP-binding cassette sub-family B member 1 ( ABCB1) are involved in the metabolism and transportation of efavirenz. The study was aimed to investigate the effects of rifampicin, CYP2B6 and ABCB1 polymorphisms on efavirenz exposure in Chinese PLWH co-infected with TB. Method PLWH were screened according to inclusion and exclusion criteria and divided into HIV group and HIV/TB group. Efavirenz plasma concentration (C0) was determined, dose-adjusted concentration (C0/D) was calculated, and genotypes of CYP2B6 516G>T, 785A>G, and ABCB1 2677G>T, 3435C>T were analyzed. Results 252 PLWH were enrolled, including 75 co-infected with TB and concomitant with rifampicin. Efavirenz C0 and C0/D were both higher in HIV group (1.94 μg/mL, 0.2007 (μg/ml)/(mg/kg/d)) compared with HIV/TB group (1.52 μg/mL, 0.1557 (μg/ml)/(mg/kg/d)) ( p = .001). Efavirenz C0/D was significantly higher in patients with variant genotypes of CYP2B6 516G>T and 785A>G ( p<.001), and was significantly lower in HIV/TB group compared with HIV group among patients with CYP2B6 516 GG, TT, and 785 AA, AG genotypes ( p < .05). Conclusion Efavirenz exposure is reduced by co-administration with rifampicin, and related to genetic polymorphisms of CYP2B6.