Central administration of neuropeptide S (NPS) in rodents induces arousal and prolonged wakefulness as well as anxiolytic-like effects. NPS has also been implicated in modulation of cognitive functions and energy homeostasis. Here we present a comprehensive phenotypical analysis of mice carrying a targeted mutation in the NPS receptor (NPSR) gene. NPSR knockout mice were found to exhibit reduced exploratory activity when challenged with a novel environment, which might indicate attenuated arousal. We also observed attenuated late peak wheel running activity in NPSR knockout mice, representing reduced activity during the subjective evening. These mice also displayed increased anxiety-like behaviors when compared to their wildtype littermates, although analysis of anxiety behaviors was limited by genetic background influences. Unexpectedly, NPSR knockout mice showed enhanced motor performance skills. No phenotypical differences were detected in the forced-swim test, startle habituation and pre-pulse inhibition paradigms. Together, these data indicate that the endogenous NPS system might be involved in setting or maintaining behavioral arousal thresholds and that the NPS system might have other yet undiscovered physiological functions.