Opioids are a potent class of analgesics in the management of both moderate to severe acute pain and chronic pain. Although neuronal response to opioids is well described, we are lacking a complete characterization of the diverse interplay of neurons with other cells types in response to opioids. Microglia, the immune cells of the CNS, are key targets of opioids and their response to repeated opioid exposure is implicated in the severe side effects associated with prolonged opioid use: opioid analgesic tolerance, opioid-induced hyperalgesia and opioid use disorder. In addition, increased microglial reactivity can alter analgesic response to opioids. Thus, understanding the role of microglia in response to opioids is both critical for improving the analgesic efficacy of opioids and for interfering with the negative side effects associated with prolonged opioid use. This thesis explores the contributions of the microglial P2X7 receptor and the microglial transcription factor Runx1 in morphine analgesia and in the development of adverse effects. My over-arching hypothesis is that increased microglial reactivity diminishes the analgesic potential of opioids, such as morphine. Here I show that repeated morphine causes a potentiation in microglial P2X7R function mediated by µ-receptor activation of Src kinase. Specifically, I identified tyrosine residues 382-384 on the P2X7R C-terminal domain as a critical site of phosphorylation and found that interfering with this site attenuated the development of tolerance in rats. I also show that the microglial transcription factor Runx1 regulates microglial reactivity in vitro and in vivo and that inhibition of Runx1 causes a decline in morphine analgesia. I characterized a novel strain of microglial-specific Runx1 knock-out mice and show that Runx1 deficiency causes a reduction in acute morphine analgesia and an exacerbation of opioid tolerance, hyperalgesia, and naloxone-precipitated withdrawal. Collectively, in this thesis I found that increased microglial reactivity, through variable mechanisms causes a reduction in the antinociceptive response to morphine in rodents. In conclusion, the work of this thesis has identified novel targets (P2X7R and Runx1) for interfering with the side effects associated with prolonged opioid use and in the acute analgesic response.