Facilitative urea transporters in the mammalian kidney play a vital role in the urinary concentrating mechanism. The urea transporters located in the renal inner medullary collecting duct, namely UT-A1 and UT-A3, are acutely regulated by the antidiuretic hormone vasopressin. In this study, we have investigated the vasopressin regulation of the basolateral urea transporter UT-A3 using an MDCK-mUT-A3 cell line. Within 10 minutes, vasopressin stimulates urea flux through UT-A3 transporters already present at the plasma membrane, via a PKA-dependent process. Within one hour, vasopressin significantly increases UT-A3 localization at the basolateral membrane, causing a further increase in urea transport. While the basic trafficking of UT-A3 to basolateral membranes involves both protein kinase C and calmodulin, its regulation by vasopressin specifically occurs through a casein kinase II-dependent pathway. In conclusion, this study details the effects of vasopressin on UT-A3 urea transporter function and hence its role in regulating urea permeability within the renal inner medullary collecting duct.