Multiple studies demonstrate that co-administration of NMDA receptor antagonists with morphine attenuates the development of analgesic tolerance. Sex differences in the effects of non-competitive, but not competitive NMDA receptor antagonists on acute morphine analgesia have been reported in mice, yet the role of sex in modulation of morphine tolerance by NMDA receptor antagonists has yet to be addressed. Therefore, we tested whether there is a sex difference in the effect of NMDA receptor antagonists on the development of morphine analgesic tolerance in C57BL/6J mice. Acutely, at a dose required to affect morphine tolerance in male mice, the non-competitive NMDA receptor antagonist MK-801 prolonged morphine analgesia similarly in both sexes in the hot plate and tail withdrawal assays. In the hot plate assay, co-administration of MK-801 or the competitive antagonist CPP [3-(2-carboxpiperazin-4-yl)propyl-1-phospionic acid] with morphine attenuated the development of tolerance in male mice, while having no effect in females. Like normal and sham females, ovariectomized mice were similarly insensitive to the attenuation of morphine tolerance by MK-801 in the hot plate assay. Surprisingly, in the tail withdrawal assay, MK-801 facilitated the development of morphine-induced hyperalgesia and tolerance in males but not females. The results demonstrate that male mice are more sensitive to modulation of nociception and morphine analgesia following repeated co-administration of NMDA receptor antagonists with morphine but that the underlying mechanisms are likely to be different from those mediating the sex difference in the modulation of acute morphine analgesia that has previously been reported.