Birds are uricotelic and, like humans, maintain high plasma urate concentrations (~300 µM). The majority of their urate waste, as in humans, is eliminated by renal proximal tubular secretion; however, the mechanism of urate transport across the brush border membrane (BBM) of the intact proximal tubule epithelium during secretion is uncertain. The dominance of secretory urate transport in the bird provides a convenient model for examining this process. The present study shows that short hairpin RNA interference effectively knocked down gene expression of Mrp4 (25% of control) in primary monolayer cultures of isolated chicken proximal tubule epithelial cells (cPTCs). Control and Mrp4-shRNAi treated cPTCs were mounted in Ussing chambers and unidirectional transepithelial fluxes of urate were measured. To detect non-specific effects, transepithelial electrical resistance (TER) and sodium-dependent glucose transport (I_glu) were monitored throughout experiments. Knocking down Mrp4 expression resulted in a reduction of transepithelial urate secretion to 35% of control with no effects on TER or I_glu. Although electrical gradient-driven urate transport in isolated BBM vesicles was confirmed, potassium-induced depolarization of the plasma membrane in intact cPTCs failed to inhibit active transepithelial urate secretion. However, electrical-gradient dependent vesicular urate transport was inhibited by the MRP4 inhibitor, MK-571, also known to inhibit active transepithelial urate transport by cPTCs. Based on these data, direct measure of active transepithelial urate secretion in functional avian proximal tubule epithelium indicates that Mrp4 is the dominant apical membrane exit pathway from cell-to-lumen.