Opioids, although fundamental to the treatment of pain, are limited in efficacy by side effects including tolerance and hyperalgesia. Using an in vitro culture system, we report that morphine increased microglial migration via a novel interaction between μ-opioid and P2X₄ receptors, which is dependent upon PI3K/Akt pathway activation. Morphine at 100 nm enhanced migration of primary microglial cells toward adenosine diphosphate by 257, 247, 301, 394, and 345% following 2, 6, 12, 24, and 48 h of stimulation, respectively. This opioid-dependent migration effect was inhibited by naloxone and confirmed to be μ-opioid receptor-dependent through the use of selective agonists and antagonists. PPADS [pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid)], a P2X_1–3,5–7 antagonist, had no effect on microglial migration; however, TNP-ATP [2′,3′-O-(2,4,6-trinitrophenyl)-ATP], a P2X_1–7 antagonist, inhibited morphine-induced migration, suggesting a P2X₄ receptor-mediated effect. The PI3K inhibitors wortmannin and LY294002 decreased morphine-induced microglial migration. Iba1 protein, a microglial marker, and P2X₄ receptor expression were significantly increased after 6, 12, 24, and 48 h of morphine stimulation. Together, these results provide evidence for two phases of morphine effects on microglia. The initial phase takes place in minutes, involves PI3K/Akt pathway activation and leads to acutely enhanced migration. The longer-term phase occurs on the order of hours and involves increased expression of Iba1 and P2X₄ receptor protein, which imparts a promigratory phenotype and is correlated with even greater migration. These data provide the first necessary step in supporting microglial migration as an attractive target for the prevention or attenuation of morphine-induced side effects including tolerance and hyperalgesia.