Regulation of urea transporter UT-A1 in the kidney is important for the urinary concentrating mechanism. We previously reported that activation of cAMP/PKA pathway by forskolin (FSK) leads to UT-A1 ubiquitination, endocytosis, and degradation. In this study, we discovered that the FSK-induced UT-A1 ubiquitination is monoubiquitination as judged by immunoblotting with specific ubiquitin antibodies to the different linkage of ubiquitin chain. The UT-A1 monoubiquitination induced by FSK was processed mainly on the cell plasma membrane. Monoubiquitination facilitates UT-A1 endocytosis and the internalized UT-A1 is accumulated in the early endosome. Inhibition of ubiquitination by E1 ubiquitin activating enzyme inhibitor PYR-41 significantly reduced FSK-induced UT-A1 endocytosis and degradation. Interestingly, FSK- stimulated UT-A1 degradation is through lysosomal protein degradation system. We further found that the PKA phosphorylation sites of UT-A1 at S486 and S499 are required for FSK induced UT-A1 monoubiquitination. The physiological significance was confirmed using rat kidney IMCD suspensions, showing that vasopressin treatment promotes UT-A1 ubiquitination. We concluded that, unlike under the basal condition that UT-A1 is subject to polyubiquitonation and proteasome-mediated protein degradation, activation of UT-A1 by FSK induces UT-A1 monoubiquitination and protein lysosomal degradation.