This study investigates the effect of electromagnetic field (EMF) on the expression of P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP) on human brain-microvascular endothelial cells (HBMECs). A monolayer of HBMECs was cocultured with human astrocytes for developing apical/basolateral membrane properties. The results revealed that an increase in the frequency of an EMF enhanced the endocytotic uptake of calcein-AM. Probenecid demonstrated a stronger inhibition to the efflux pumps than verapamil under the exposure to an EMF. The expressed quantities of P-gp and MRP1 after transcytosis of saquinavir were higher than those of azidothymidine, suggesting that the former induced a stronger drug resistance than the latter. In addition, an EMF with 750Hz could effectively inhibit the expression of P-gp and MRP1. An appropriate exposure to an EMF can be efficient to deliver antiretroviral drugs into the brain via reducing the expressed efflux transporters on HBMECs.