P-glycoprotein (P-gp) is involved in the ATP-dependant cellular efflux of a large number of drugs including ivermectin, a macrocyclic lactone (ML) endectocide, widely used in livestock and human antiparasitic therapy. The interactions of P-gp with ivermectin and other MLs were studied. In a first approach, the ability of ivermectin (IVM), eprinomectin (EPR), abamectin (ABA), doramectin (DOR), selamectin (SEL), or moxidectin (MOX) to inhibit the rhodamine123 efflux was measured in recombinant cells overexpressing P-gp. Then, the influence of these compounds on the P-gp ATPase activity was tested on membrane vesicles prepared from fibroblasts overexpressing P-gp. All the MLs tested increased the intracellular rhodamine123. However, the potency of MOX to inhibit P-gp function was 10 times lower than the other MLs. They all inhibited the basal and decreased the verapamil-stimulated P-gp ATPase activity. But SEL and MOX were less potent than the other MLs when competing with verapamil. According to the structural specificity of SEL and MOX, we conclude that the integrity of the sugar moiety is determinant to achieve the optimal interaction of macrocyclic lactones with P-gp. The structure-affinity relationship for interaction with P-gp is important information for improving ML bioavailability and reversal of multidrug resistance (MDR).