KOR agonists possess adverse dysphoric and psychotomimetic effects, thus limiting their applications as anti-pruritic and non-addictive analgesic agents. Here, we showed that protein kinase C (PKC) inhibition preserved the KOR agonist induced beneficial antinociceptive and antipruritic effects, and attenuated the adverse condition placed aversion (CPA), sedation, and motor incoordination in mice. Using a large-scale mass spectrometry-based phosphoproteomics of KOR-mediated signaling in the brain, we observed at 5 minutes PKC-dependent modulation of G protein-coupled receptor kinases and Wnt pathways, and at 30 minutes modulation of stress signaling, cytoskeleton, mTOR signaling and receptor phosphorylation, including cannabinoid receptor CB1. We further demonstrated that inhibition of CB1 attenuated KOR-mediated CPA. Our results demonstrated the feasibility of in vivo biochemical dissection of signaling pathways that lead to side effects.