Sex differences in the experience of clinical and experimental pain have been reported. However, the neurobiological sources underlying the variability in pain responses between sexes have not been adequately explored, especially in humans. The endogenous opioid neurotransmitters and mu-opioid receptors are centrally implicated in responses to stress, in the suppression of pain, and in the action of opiate analgesic drugs. Here we examined sex differences in the activation of the mu-opioid system in response to an intensity-controlled sustained deep-tissue pain challenge with positron emission tomography and a mu-opioid receptor-selective radiotracer. Twenty-eight young healthy volunteers (14 men and 14 women) were studied during saline control and pain conditions using a double-blind, randomized, and counterbalanced design. Women were scanned during the early follicular phase of their menstrual cycles after ovulatory cycles. Significant sex differences in the regional activation of the mu-opioid system in response to sustained pain were detected compared with saline controls. Men demonstrated larger magnitudes of mu-opioid system activation than women in the anterior thalamus, ventral basal ganglia, and amygdala. Conversely, women demonstrated reductions in the basal state of activation of the mu-opioid system during pain in the nucleus accumbens, an area previously associated with hyperalgesic responses to the blockade of opioid receptors in experimental animals. These data demonstrate that at matched levels of pain intensity, men and women during their follicular phase differ in the magnitude and direction of response of the mu-opioid system in distinct brain nuclei.