Efflux pumps, P-glycoprotein (P-gp), multidrug resistance-associated proteins (MRPs), and breast cancer resistance protein (BCRP) have been shown to extrude HIV protease inhibitors from cells. These transporters are present on many barrier sites such as the blood-brain barrier (BBB) and on many circulating cells such as lymphocytes, and could reduce protease inhibitor concentration in sanctuary or HIV-1 target sites. This study compares the potential of the antiretroviral drug atazanavir to modulate P-gp and MRP expression and function in total lymphocytes and in human fetal brain endothelial cells (HBMECs). We address the question of atazanavir transport across the human BBB. Following incubation with atazanavir, P-gp and MRP1 expression was determined by direct immunofluorescence. Transporter function was assessed by measuring fluorescent dye efflux, either with or without specific inhibitors. Atazanavir substrate properties were determined by transport quantification through a validated in vitro human BBB model. Our results show that in contrast to HBMECs, in lymphocytes, atazanavir has no effect on MRP1 and P-gp expression. However, there were overall changes in P-gp function increasing its activity in lymphocytes and HBMECs. Using the in vitro human BBB model, we confirm the interaction of atazanavir with P-gp, MRPs, and BCRP in preventing its passage across this barrier and thus its entry into the central nervous system.