Bitter taste, in humans, is sensed by 25 G protein-coupled receptors, referred to as bitter taste receptors (T2Rs). The diverse roles of T2Rs in various extraoral tissues have implicated them as a potential target for therapeutic intervention. Structure-function studies have provided insights into the role of transmembrane and loop regions in the activation mechanism of T2Rs. However, studies aimed at deciphering the role of their carboxyl-terminus (C-terminus) are limited. In this study, we identified a KLK/R motif in the C-terminus that is conserved in 19 of the 25 T2Rs. Using site-directed mutagenesis we studied the role of 16 residues in the C-terminus of T2R4. The C-terminus of T2R4 is polybasic with 6 of the 16 residues consisting of lysines, constituting two separate KK motifs. We analyzed the effect of the C-terminus mutations on plasma membrane trafficking, and characterized their function in response to the T2R4 agonist quinine. The majority of the mutants showed defective receptor trafficking with ≤50% expression on the cell surface. Interestingly, mutation of the distal Lys296 of the KLK motif in T2R4 resulted in constitutive activity. The K296A mutant displayed five-fold basal activity over wild type T2R4, while the conservative substitution K296R showed wild type characteristics. The Lys294, Leu295 and Lys296 of the KLK motif in T2R4 were found to perform crucial roles, both, in receptor trafficking and function. Results from this study provide unique mechanistic insights into the structure-function role of the C-terminus in T2R signaling.