Coordination of cell and tissue polarity commonly involves directional signaling. In the Arabidopsis root epidermis, cell polarity is revealed by basal, root tip-oriented, hair outgrowth from hair-forming cells (trichoblasts). The plant hormone auxin displays polar movements and accumulates at maximum concentration in the root tip. The application of polar auxin transport inhibitors evokes changes in trichoblast polarity only at high concentrations and after long-term application. Thus, it remains open whether components of the auxin transport machinery mediate establishment of trichoblast polarity. Here we report that the presumptive auxin influx carrier AUX1 contributes to apical-basal hair cell polarity. AUX1 function is required for polarity changes induced by exogenous application of the auxin 2,4-D, a preferential influx carrier substrate. Similar to aux1 mutants, the vesicle trafficking inhibitor brefeldin A (BFA) interferes with polar hair initiation, and AUX1 function is required for BFA-mediated polarity changes. Consistently, BFA inhibits membrane trafficking of AUX1, trichoblast hyperpolarization induced by 2,4-D, and alters the distal auxin maximum. Our results identify AUX1 as one component of a novel BFA-sensitive auxin transport pathway polarizing cells toward a hormone maximum.