Nitric oxide signalling has been implicated in impulsive and aggressive traits and behaviours in both animals and humans. In the present study, we investigated the effects of a functional variable number of tandem repeats (VNTR) polymorphism in exon 1f (ex1f) of the nitric oxide synthase 1 (NOS1) gene (NOS1 ex1f-VNTR) and stressful life events on aggressive behaviour in population representative sample of adolescents followed up from third grade to 25 years of age. We studied the younger cohort of the longitudinal Estonian Children Personality, Behaviour and Health Study (subjects in the last study wave n = 437, males n = 193; mean age 24.8 ± 0.5 years). Aggressive behaviour was rated at age 25 with the Illinois Bully Scale and Buss-Perry Aggression Questionnaire. Life history of aggression was evaluated in a structured interview. Stressful life events and family relationships were self-reported at age 15. The hypothesized risk genotype (homozygosity for the short allele) was associated with higher levels of aggression in males (statistical significance withstanding the multiple correction procedure). Exposure to stressful life events or adverse family relationships was associated with increased aggressive behaviour in subjects homozygous for either of the alleles, and these associations were mostly observed in males. However, these associations in these stratified analyses did not survive correction for multiple testing. Aggressiveness was relatively unaffected by the NOS1 ex1f-VNTR genotype in the female subjects even when taking exposure to childhood adversity into account. Our findings support the hypothesized involvement of a functional NOS1 polymorphism on aggression in a population representative sample of young adults.