The opioid receptors are a member of G protein-coupled receptors that mediate physiological effects of endogenous opioid peptides and structurally distinct opioid alkaloids. Although it is well characterized that there is differential receptor desensitization and internalization properties following activation by distinct agonists, the underlying mechanisms remain elusive. We investigated the signaling events of delta-opioid receptor (deltaOR) initiated by two ligands, DPDPE and TIPP. We found that although both ligands inhibited adenylyl cyclase (AC) and activated ERK1/2, only DPDPE induced desensitization and internalization of the deltaOR. We further found that DPDPE, instead of TIPP, could activate GRK2 by phosphorylating the non-receptor tyrosine kinase Src and translocating it to membrane receptors. Activation of GRK2 led to the phosphorylation of serine residues in the C-terminal tail, which facilitates beta-arrestin1/2 membrane translocation. Meanwhile, we also found that DPDPE promoted beta-arrestin1 dephosphorylation in a Src-dependent manner. Thus, DPDPE appears to strengthen beta-arrestin function by dual regulations: promoting beta-arrestin recruitment and increasing beta-arrestin dephosphorylation at the plasma membrane in a Src-dependent manner. All effects initiated by DPDPE could be abolished or suppressed by PP2, an inhibitor of Src. Morphine, which has been previously shown to be unable to desensitize or internalize deltaOR, also behaved as TIPP in failure to utilize Src to regulate deltaOR signaling. These findings point to the existence of agonist-specific utilization of Src to regulate deltaOR signaling and reveal the molecular events by which Src modulates deltaOR responsiveness.