Improving rehabilitation after a severe tissue injury does not only require a reduction in pain, but also requires alleviation of negative affects, particularly anxiety. Although opioids remain unsurpassed analgesics to relieve moderate to severe pain, it has been shown that they also induce latent pain sensitization leading to long-lasting hyperalgesia via N-methyl-D-aspartate-(NMDA)-dependent pronociceptive systems. The present study evaluated the ability of nitrous oxide (N2O), a gas with NMDA antagonist properties, to prevent latent pain sensitization and long-term anxiety-like behavior (ALB) in rats with pain and opioid experiences. On D0, the pro-inflammatory drug carrageenan was injected in one hind paw of rats treated with fentanyl (4x100 microg/kg subcutaneously). Nociceptive threshold was evaluated with the paw pressure vocalization test. Rats were re-exposed to carrageenan or exposed to repeated non-nociceptive environmental stress (NNES) 2-3 weeks later. Rats were also challenged in the elevated plus-maze 2 weeks after fentanyl administration for evaluating ALB. The preventive effects of a single 4 h 50/50% N2O-O2 exposure performed on D0 was evaluated. Fifty percent N2O strongly reduced hyperalgesia induced by a first inflammation and its enhancement by fentanyl, and prevented exaggerated hyperalgesia induced by second inflammatory pain or NNES. Moreover, we provide first evidence that a high fentanyl dose induces long-term ALB 2 weeks after its administration. When associated with fentanyl, 50% N2O prevented such long-term ALB. These results suggest that a single exposure to N2O could improve post-injury pain management and facilitate rehabilitation especially when potent analgesics as opioids have to be used.