Although opioids still remain the most powerful pain-killers, the chronic use of opioid analgesics is largely limited by their numerous side-effects, including opioid dependence. However, the mechanism underlying this dependence is largely unknown. In this study, we used the withdrawal symptoms precipitated by naloxone to characterize opioid dependence in mice. We determined the functional role of mu-opioid receptors (MORs) expressed in different subpopulations of neurons in the development of morphine withdrawal. We found that conditional deletion of MORs from glutamatergic neurons expressing vesicular glutamate transporter 2 (Vglut2) largely eliminated the naloxone-precipitated withdrawal symptoms. In contrast, conditional deletion of MORs expressed in GABAergic neurons had a limited effect on morphine withdrawal. Consistently, mice with MORs deleted from Vglut2 glutamatergic neurons also showed no morphine-induced locomotor hyperactivity. Furthermore, morphine withdrawal and morphine-induced hyperactivity were not significantly affected by conditional knockout of MORs from dorsal spinal neurons. Taken together, our data indicate that the development of morphine withdrawal is largely mediated by MORs expressed in Vglut2 glutamatergic neurons.