Nutritional supplementation to TB patients has been associated with increase weight and mortality reduction, but its effect on the pharmacokinetics of first-line anti-TB drugs is unknown. A cohort of 100 TB patients (58 men, median age 35 (IQR: 29; 40) years and BMI 18.8 (17.3; 19.9) kg/m(2)) were randomized to receiving nutritional supplementation during the intensive phase of TB treatment. Rifampicin plasma concentrations were determined after 1 week and 2 months of treatment. The effects of nutritional supplementation, HIV, time on treatment, body weight, and SLCO1B1 rs4149032 genotype were examined using a population pharmacokinetic model. The model adjusted for body size via allometric scaling, accounted for clearance auto-induction, and detected an increase in bioavailability (+14%) for the patients in the continuation phase. HIV co-infection in patients not receiving the supplementation was found to decrease bioavailability by 21.8%, with a median Cmax and AUC0-24h of 5.6 μg/mL and 28.6 μg⋅h/mL, respectively. HIV co-infected on nutritional supplementation achieved higher Cmax and AUC0-24h of 6.4 μg/mL and 31.6 μg⋅h/mL respectively and only 13.3% bioavailability reduction. No effect of the SLCO1B1 rs4149032 genotype was observed. In conclusion, nutritional supplementation during the first 2 months of TB treatment reduces the decrease in rifampicin exposure observed in HIV co-infected patients, but does not affect exposure in HIV uninfected. If confirmed in other studies, the use of defined nutritional supplementation in HIV co-infected TB patients should be considered in TB control programs.