Opioids and other agonists of the μ-opioid receptor are effective at managing acute pain, but their chronic use can lead to tolerance that limits their efficacy. We previously reported that inhibiting the chaperone protein HSP90 in the spinal cords of mice promotes the antinociceptive effects of opioids in a manner that involved increased activation of the kinase ERK. Here, we found that the underlying mechanism involves the relief of a negative feedback loop mediated by the kinase AMPK. Intrathecal treatment of male and female mice with the HSP90 inhibitor 17-AAG decreased the abundance of the β1 subunit of AMPK in the spinal cord. The antinociceptive effects of 17-AAG with morphine were suppressed by intrathecal administration of AMPK activators and enhanced by an AMPK inhibitor. Opioid treatment increased the abundance of phosphorylated AMPK in the dorsal horn of the spinal cord, where it colocalized with a neuronal marker and the neuropeptide CGRP. Knocking down AMPK in CGRP-positive neurons enhanced the antinociceptive effects of morphine and demonstrated that AMPK mediated the signal transduction between HSP90 inhibition and ERK activation. These data suggest that AMPK mediates an opioid-induced negative feedback loop in CGRP neurons of the spinal cord and that this loop can be disabled by HSP90 inhibition to enhance the efficacy of opioids.