Neuropeptide S (NPS), a recently identified bioactive peptide through reverse pharmacology approach, was reported to regulate arousal, anxiety, locomotor activity, feeding behaviors and drug reward. NPS receptor (NPSR) mRNA was found in the area related to the descending control system of pain, such as the periaqueductal gray (PAG), raphe nuclei, and lateral parabrachial nucleus (PBN), suggesting a possible role of the NPS-NPSR system in the regulation of pain transmission. In the present study, we evaluated the effects of NPS in pain modulation at the supraspinal level for the first time, using the tail withdrawal test and hot-plate test in mice. NPS (mouse, 0.01-1 nmol) injected intracerebroventricularly (i.c.v.) caused a significant increase of tail withdrawal latency and paw-licking/jumping latency in the tail withdrawal test and the hot-plate test, respectively. Antinociceptive effect elicited by NPS (0.1 nmol, i.c.v.) was not affected by naloxone (i.c.v., 10 nmol co-injection or i.p., 10 mg/kg, 10 min prior to NPS) in both tail withdrawal test and hot-plate test. However, at the doses, naloxone significantly inhibited the antinociceptive effect induced by morphine (i.c.v., 3 nmol). NPS (0.1 nmol, i.c.v.)-induced antinociception was inhibited by co-injection with 10 nmol, but not 3 nmol [D-Cys(tBu)(5)]NPS, a peptidergic antagonist identified more recently, while [D-Cys(tBu)(5)]NPS (3 and 10 nmol) alone induced neither hyperalgesia nor antinociception. These results revealed that NPS could produce antinociception through NPS receptor, but not opioid receptor, and NPS-NPSR system could be a potential target for developing new analgesic drugs.