Withdrawal from systemic opioids can induce long-term potentiation (LTP) at spinal C-fibre synapses ("opioid-withdrawal-LTP"). This is considered to be a cellular mechanism underlying opioid withdrawal-induced hyperalgesia, which is a major symptom of the opioid withdrawal syndrome. Opioids can activate glial cells leading to the release of pro-inflammatory mediators. These may influence synaptic plasticity and could thus contribute to opioid-withdrawal-LTP. Here, we report a sexual dimorphism in the mechanisms of morphine-withdrawal-LTP in adult rats.We recorded C-fibre-evoked field potentials in the spinal cord dorsal horn from deeply anaesthetised male and female rats. In both sexes we induced a robust LTP via withdrawal from systemic morphine infusion (8 mg·kg bolus, followed by a one-hour infusion at a rate of 14 mg·kg·h). This paradigm also induced mechanical hypersensitivity of similar magnitude in both sexes. In male rats, systemic but not spinal application of (-)naloxone blocked the induction of morphine-withdrawal-LTP, suggesting the involvement of descending pro-nociceptive pathways. Furthermore, we showed that in male rats, the induction of morphine-withdrawal-LTP required the activation of spinal astrocytes and the release of the pro-inflammatory cytokines tumour necrosis factor (TNF) and interleukin-1 (Il-1). In striking contrast, in female rats, the induction of morphine-withdrawal-LTP was independent of spinal glial cells. Instead, blocking µ-opioid receptors in the spinal cord was sufficient to prevent a facilitation of synaptic strength.Our study revealed fundamental sex differences in the mechanisms underlying morphine-withdrawal-LTP at C-fibre synapses: supraspinal and gliogenic mechanisms in males and a spinal, glial cell-independent mechanism in females.