Protein phosphorylation is a key posttranslational modification mechanism controlling the conformation and activity of many proteins. Increasing evidence has implicated an essential role of phosphorylation by several major protein kinases in promoting and maintaining opioid tolerance. We review some of the most recent studies on protein kinase C (PKC), cyclic AMP dependent protein kinase A (PKA), calcium/calmodulin-dependent protein kinase II (CaMKII), protein kinase G (PKG), and G protein receptor kinase (GRK). These kinases act as the molecular switches to modulate opioid tolerance. Pharmacological interventions at one or more of the protein kinases and phosphatases may provide valuable strategies to improve opioid analgesia by attenuating tolerance to these drugs.