Neuropeptide S is involved in anxiety and arousal modulation, and the functional polymorphism Asn107Ile (rs324981, A > T) of the neuropeptide S receptor gene (NPSR1) is associated with panic disorder and anxiety/fear-related traits. NPSR1 also interacts with the environment in shaping personality and impulsivity. We therefore examined whether the NPSR1 A/T polymorphism is associated with affective and anxiety disorders in a population-representative sample. Lifetime psychiatric disorders were assessed by MINI interview (n = 501) in the older cohort of the longitudinal Estonian Children Personality, Behaviour and Health Study (ECPBHS). Anxiety (STAI), self-esteem (RSES), depression (MÅDRS), suicide attempts and environmental factors were self-reported in both the younger (original n = 583) and the older cohort (original n = 593). Most of the NPSR1 effects were sex-specific and depended on environmental factors. Females with the functionally least active NPSR1 AA genotype and exposed to environmental adversity had affective/anxiety disorders more frequently; they also exhibited higher anxiety and depressiveness, and lower self-esteem. Female AA homozygotes also reported suicidal behaviour more frequently, and this was further accentuated by adverse family environment. In the general population, the NPSR1 A/T polymorphism together with environmental factors is associated with anxious, depressive and activity-related traits, increased prevalence of affective/anxiety disorders and a higher likelihood of suicidal behaviour.