The altered behavioral effects of morphine, but not most other mu agonists, in mice lacking β-arrestin 2, suggest that this scaffolding protein regulates the signaling cascade of this commonly used analgesic. One of the cascades that could be regulated by β-arrestin 2 is cJun-N-terminal kinase (JNK), which binds with β-arrestin 2 and modulates the analgesic effects of morphine. Using neurons lacking β-arrestin 2 (β-arr2−/−) to examine this interaction, we found that β-arr2−/− neurons show altered intracellular distribution of JNK and cJun, and that morphine, but not fentanyl, increased the nuclear localization of the phosphorylated, therefore activated, form of cJun, a JNK target in dorsal root ganglia neurons. This suggests that deleting β-arrestin 2 affects the JNK cascade. We therefore examined whether some of the behavioral phenotypes of mice lacking β-arrestin 2 could be a result of altered JNK signaling. Indeed, two different JNK inhibitors reversed the enhanced analgesic effect of morphine, a known phenotype of β-arr2−/− mice, to +/+ levels. Both the reduced locomotor effect of morphine and the psychomotor sensitization to repeated morphine administration in β-arr2−/− mice were also returned to +/+ levels by inhibiting JNK. In contrast, the behavioral effects of fentanyl were neither genotype-dependent nor affected by JNK inhibition. Furthermore, a PKC inhibitor had a similar effect as inhibiting JNK in reducing the enhanced analgesic effect of morphine in β-arr2−/− mice to +/+ levels. In summary, removing β-arrestin 2 reveals mu receptor activation of the JNK cascade in a ligand-specific manner explaining several behavioral phenotypes of β-arr2−/− mice.