Abstract We have demonstrated that the activation of p38 mitogen-activated protein kinase (MAPK) in the spinal microglia played an essential role in the development of morphine antinociceptive tolerance. The aim of this study was to investigate whether inhibition of neuronal nitric oxide synthase (nNOS) attenuated tolerance to morphine analgesia by modulating p38 activation in the spinal microglia. It was shown that the selective inhibitor of nNOS, 7-NINA (7-Nitroindazole, sodium salt) (25 μg, i.t.) attenuated not only the development of morphine antinociceptive tolerance, but also the activation of p38 MAPK in the spinal microglia induced by chronic intrathecal administration of morphine. Our results suggest that neuronal NO signals to microglia, leading to the upregulation of microglial phospho-p38 MAPK. Such p38 MAPK activation in microglia is consistent with a potential role in the development of morphine antinociceptive tolerance. We demonstrated for the first time that the inhibition of nNOS attenuated morphine antinociceptive tolerance by reducing p38 MAPK activation in the spinal microglia.