In the present study, the effect of chronic fatigue stress on the neuronal nitric oxide synthase (nNOS) in the rat nucleus accumbens (NAc) was assessed in order to explore the neurobiology mechanism of central fatigue stress, especially the role of the NAc in central fatigue. In the experiment, adult male Wistar rats were forced to swim till exhaustion every day for 4 weeks. Immunohistochemistry was used for measurement of the nNOS-positive neurons associated with the image manipulation. Our results showed that a long-time exposure to chronic forced swim stress increased the number of nNOS immunoreactive-positive neurons in the rat NAc (106.7%, P<0.001), distributed area (150.2%, P<0.001) and gray degree value (11.3%, P<0.01). The results indicate that nitric oxide (NO)/nNOS may be involved in the formation of the fatigue stress, and that NO may play a role in the regulation of stress in the NAc. The up-regulation of NO/nNOS during the exposure to long-time swim stress is likely to be one of the results of brain damage and psychiatric disorder induced by NO overproduction.