Human organic anion transporter 1 (hOAT1) expressed at the basolateral membrane of the kidney proximal tubule cells mediates the active renal secretion of a diverse array of clinically important drugs, including anti-HIV therapeutics, anti-tumor drugs, antibiotics, anti-hypertensives, and anti-inflammatories. We previously demonstrated that post-translational modification of hOAT1 by ubiquitination is an important mechanism for the regulation of this transporter. The current study aimed at identifying the ubiquitin ligase for hOAT1 and its mechanism of action. We showed that overexpression of Nedd4-1 (neural precursor cell expressed, developmentally down-regulated 4-1), an E3 ubiquitin ligase, enhanced hOAT1 ubiquitination, decreased hOAT1 expression at the cell surface, and inhibited hOAT1 transport activity. In contrast, overexpression of the ubiquitin ligase-dead mutant Nedd4-1/C867S was without effects on hOAT1. Furthermore, knockdown of endogenously expressed Nedd4-1 by Nedd4-1-specific siRNA reduced hOAT1 ubiquitination. Immunoprecipitation experiments in cultured cells and in rat kidney slices and immunofluorescence study in rat kidney slices showed that there was a physical interaction between OAT1 and Nedd4-1. Nedd4-1 contains four protein-protein interacting WW domains. When these WW domains were inactivated by mutating two amino acid residues in each of the four WW domains respectively (Mut-WW1: V210W/H212G; Mut-WW2: V367W/H369G; Mut-WW3: I440W/H442G; Mut-WW4: I492W/H494G), only Mut-WW2 and Mut-WW3 significantly lost their ability to bind and to ubiquitinate hOAT1. As a result, Mut-WW2 and Mut-WW3 were unable to suppress hOAT1-mediated transport as effectively as wild type Nedd4-1. In conclusion, this is the first demonstration that Nedd4-1 regulates hOAT1 ubiquitination, expression, and transport activity through its WW2 and WW3 domains.