The phytohormone auxin is critical for plant growth and many developmental processes. Members of the P-glycoprotein (PGP/ABCB) subfamily of ABC transporters have been shown to function in the polar movement of auxin by transporting auxin over the plasma membrane in both monocots and dicots. Here, we characterize a new Arabidopsis member of the ABCB subfamily, ABCB21/PGP21, a close homolog of ABCB4, for that conflicting transport directionalities have been reported. ABCB21 is strongly expressed in the abaxial side of cotyledon and in junctions of lateral organs in the aerial part, whereas in roots it was specifically expressed in pericycle cells. Membrane fractionation by sucrose density gradient centrifugation followed by western blot showed that ABCB21 is a plasma membrane-localized ABC transporter. Transport assay with Arabidopsis protoplasts suggested that ABCB21 was involved in IAA transport in an outward direction, while NAA was a less preferable substrate for ABCB21. Further functional analysis of ABCB21 using yeast im- and export assays showed that ABCB21 mediates the NPA-sensitive translocation of auxin in an inward direction when cytoplasmic IAA concentration is low, whereas this transporter mediates outward transport under high internal IAA. Increase in the cytoplasmic IAA concentration by pre-loading of IAA into yeast cells abolished the IAA uptake activity by ABCB21 as well as ABCB4. These findings suggest that ABCB21 functions as a facultative im-/exporter controlling auxin concentrations in plant cells.