Amyloid‐β (Aβ) is the major component of the insoluble amyloid plaques that accumulate intracerebrally in patients with Alzheimer’s disease (AD). It has been suggested that MDR1‐P‐glycoprotein (ABCB1, P‐gp) plays a substantial role in the elimination of Aβ from the brain. In the present study, MDR1‐transfected LLC cells growing in a polarized cell layer were used to characterize the interaction of Aβ1‐40/1‐42 with P‐gp. In this system, P‐gp‐mediated transport can be followed by the efflux of the fluorescent dye rhodamine‐123, or of Aβ itself from the cells into the apical extracellular space. Aβ significantly decreased the apical efflux of rhodamine‐123, and the transcellular transport of Aβ1‐40 and Aβ1‐42 into the apical chamber could be demonstrated using both ELISA and fluorescence (FITC)‐labeled peptides. This transport was inhibited by a P‐gp modulator. Furthermore, ATP‐dependent, P‐gp‐mediated transport of the fluorescence‐labeled peptides could be demonstrated in isolated, inside‐out membrane vesicles. Our data support the concept that P‐gp is important for the clearance of Aβ from brain, and thus may represent a target protein for the prevention and/or treatment of neurodegenerative disorders such as AD.