We reported previously that the selective agonist U50,488H promoted phosphorylation of the mouse kappa opioid receptor (KOPR) at residues S356, T357, T363 and S369. Here, we found that agonist (U50,488H)-dependent KOPR phosphorylation at all the residues were mediated by Gi/oα proteins and multiple protein kinases [GRKs2, 3, 5 and 6 and protein kinase C (PKC)]. In addition, PKC activation by phorbol ester induced agonist-independent KOPR phosphorylation. Compared with U50,488H, PKC activation promoted much higher S356/T357 phosphorylation, much lower T363 phosphorylation and similar levels of S369 phosphorylation. Following U50,488H, GRKs, but not PKC, were involved in agonist-induced KOPR internalization. In contrast, PKC activation caused a lower level of agonist-independent KOPR internalization, compared to U50,488H. U50,488H-induced activation of extracellular signal regulated kinase 1/2 (ERK1/2) was G protein-, but not β-arrestin-, dependent. After U50,488H, GRK-mediated, but not PKC-mediated, KOPR phosphorylation followed by β-arrestin recruitment desensitized U50,488H-induced ERK1/2 response. Therefore, agonist-dependent (GRK- and PKC-mediated) and agonist-independent (PKC-promoted) KOPR phosphorylations show distinct phosphorylation patterns, leading to diverse cellular outcomes.