Kappa-opioid receptor (KOR) is a member of G-protein coupled receptors (GPCRs) expressed in serotonergic neurons and neuronal terminals. The involvement of KOR ligands in nociception, diuresis, emotion, cognition, and immune system has been extensively studied. Omics-based methods are preferable to understand the signaling cascade after KOR activation in a systematic manner. In this study, an in-depth quantitative phosphoproteomic analysis resulted in 305 phosphosites, which were significantly changed in three KOR-overexpressed cells upon treatment with two KOR agonists. The subsequent substrate-kinase prediction analysis revealed that 18 potential kinases might be activated under stimulation of the agonists. We found that phosphorylation of PAK1/2 (p21-activated kinase 1/2) was induced by KOR agonists, resulting in reduced actin stress fibers and cytoskeletal reorganization. In summary, this quantitative phosphoproteomics-based research studied the downstream phosphorylation events upon KOR activation, which may shed light on the investigations of KOR signaling pathway and targeted therapy for KOR-related diseases. (C) 2019 Elsevier Inc. All rights reserved.