Protein kinase C (PKC) activation plays an important role in morphine-induced μ-opioid receptor (OPRM1) desensitization and tolerance development. It was recently shown that receptor phosphorylation by G protein-coupled receptor kinase regulates agonist-dependent selective signaling and that inefficient phosphorylation of OPRM1 leads to PKCε activation and subsequent responses. Here, we demonstrate that such receptor phosphorylation and PKCε activation can be modulated by FK506-binding protein 12 (FKBP12). Using a yeast two-hybrid screen, FKBP12 was identified as specifically interacting with OPRM1 at the Pro(353) residue. In human embryonic kidney 293 cells expressing OPRM1, the association of FKBP12 with OPRM1 decreased the agonist-induced receptor phosphorylation at Ser(375). The morphine-induced PKCε activation and the recruitment of PKCε to the OPRM1 signaling complex were attenuated both by FKBP12 short interfering RNA (siRNA) treatment and in cells expressing OPRM1 with a P353A mutation (OPRM1P353A), which leads to diminished activation of PKC-dependent extracellular signal-regulated kinases. Meanwhile, the overexpression of FKBP12 enabled etorphine to activate PKCε. Further analysis of the receptor complex demonstrated that morphine treatment enhanced the association of FKBP12 and calcineurin with the receptor. The blockade of the FKBP12 association with the receptor by the siRNA-mediated knockdown of endogenous FKBP12 or the mutation of Pro(353) to Ala resulted in a reduction in PKCε and calcineurin recruitment to the receptor signaling complex. The receptor-associated calcineurin modulates OPRM1 phosphorylation, as demonstrated by the ability of the calcineurin autoinhibitory peptide to increase the receptor phosphorylation. Thus, the association of FKBP12 with OPRM1 attenuates the phosphorylation of the receptor and triggers the recruitment and activation of PKCε.