There is an increased recognition of the need to identify and quantify the impact of genetic polymorphisms on drug-drug interactions. This study investigated the pharmacogenetics of the pharmacokinetic drug-drug interaction between nevirapine and artemether-lumefantrine in HIV-positive and HIV-negative adult Nigerian subjects. Thirty each of HIV-infected patients on nevirapine-based antiretroviral therapy and HIV-negative volunteers without clinical malaria, but with predetermined CYP2B6 c.516GG and TT genotypes were administered a complete treatment dose of 3-days of artemether-lumefantrine. Rich pharmacokinetic sampling prior to and following the last dose was conducted and plasma concentrations of artemether/dihydroartemisinin and, lumefantrine/desbutyl-lumefantrine were quantified using tandem mass spectrometry. Pharmacokinetic parameters of artemether-lumefantrine and its metabolites in HIV-infected patients on nevirapine were compared with those in the absence of nevirapine in HIV-negative volunteers. Overall, nevirapine reduced exposure to artemether and desbutyl-lumefantrine by 39% and 34% respectively. These reductions were significantly greater in GG versus TT subjects for artemether [ratio of geometric mean (90% confidence interval): 0.42 (0.29 – 0.61) versus 0.81 (0.51 – 1.28)] and for desbutyl-lumefantrine [0.56 (0.43 – 0.74) versus 0.75 (0.56 – 1.00)]. On the contrary, it increased exposure to dihydroartemisinin and lumefantrine by 47% and 30% respectively. These increases were significantly higher in TT versus GG subjects for dihydroartemisinin [1.67 (1.20 – 2.34) versus 1.25 (0.88 – 1.78)] and for lumefantrine [1.51 (1.20 – 1.90) versus 1.08 (0.82 – 1.42)]. This study underscores the importance of incorporating pharmacogenetics into all drug-drug interaction studies with potential for genetic polymorphisms to influence drug disposition.