Activated human neuropeptide Y Y1 receptors rapidly desensitize and internalize through clathrin-coated pits and recycle from early and recycling endosomes, unlike Y2 receptors that neither internalize nor desensitize. To identify motifs implicated in Y1 receptor desensitization and trafficking, mutants with varying C-terminal truncations or a substituted Y2 C-terminus were constructed. Point mutations of key putative residues were made in a C-terminal conserved motif [φ-H-(S/T)-(E/D)-V-(S/T)-X-T] that we have identified and in the second intracellular i2 loop. Receptors were analyzed by functional assays, spectrofluorimetric measurements on living cells, flow cytometry, confocal imaging and bioluminescence resonance energy transfer assays for β-arrestin activation and adaptor protein (AP-2) complex recruitment. Inhibitory GTP-binding protein-dependent signaling of Y1 receptors to adenylyl cyclase and desensitization was unaffected by C-terminal truncations or mutations, while C-terminal deletion mutants of 42 and 61 amino acids no longer internalized. Substitutions of Thr357, Asp358, Ser360 and Thr362 by Ala in the C-terminus abolished both internalization and β-arrestin activation but not desensitization. A Pro145 substitution by His in an i2 consensus motif reported to mediate phosphorylation-independent recruitment of β-arrestins affected neither desensitization, internalization or recycling kinetics of activated Y1 receptors nor β-arrestin activation. Interestingly, combining Pro145 substitution by His and C-terminal substitutions significantly attenuates Y1 desensitization. In the Y2 receptor, replacement of His155 with Pro at this position in the i2 loop motif promotes agonist-mediated desensitization, β-arrestin activation, internalization and recycling. Overall, our results indicate that β-arrestin-mediated desensitization and internalization of Y1 and Y2 receptors are differentially regulated by the C-terminal motif and the i2 loop consensus motif.