At least 45% of prisoners in Australia are incarcerated for violent offences. Nutritional intervention trials, including omega-3 fatty acids, have shown promising results in reducing aggressive behaviour in prisoners, however, blood biometrics and the Inmate Behavioural Observation Score (IBOS), a measure of aggressive behaviour in custody that has construct validity across jurisdictions(1), were lacking. The aim of this study was to determine whether omega-3 supplementation is effective in reducing aggressive behaviour in adult male prisoners using a randomised, double-blind, placebo-controlled intervention. Ethical approval was obtained from the University of Wollongong Human Research Ethics Committee (HREC) (2017/163), Corrective Services New South Wales (NSW) Ethics Committee (D17.847215), the NSW Justice Health & Forensic Mental Health Network HREC (G310/17), and the Aboriginal Health & Medical Research Council of NSW Ethics Committee (1321/17). Adult male prisoners (n = 314) with a baseline IBOS >0 (indicating some level of aggressive behaviour) and erythrocyte omega-3 index <6% were recruited and block randomised according to their IBOS, age and baseline omega-3 index, within each treatment centre to receive either 1 g omega-3/day or placebo. The IBOS data were analyzed using linear mixed models with centre as a random term to adjust for clustering. IBOS was analyzed as the score and the cube root of the score to improve normality. P values were also estimated using parametric bootstraps. Baseline and 16-week post randomisation assessments included primary outcome measure – the IBOS, and secondary outcome measures: Aggression Questionnaire; Institutional Records of Misconduct; Questionnaires – Attention Deficit Disorders; Attention Deficit Hyperactivity Disorders; Depression Anxiety and Stress Scale; Barratt Impulsiveness Scale score; Quality of Life; sleep; muscle strength; and engagement in, and outcomes of, rehabilitation programs. Of the approximately 6,600 prisoners in the 7 participating Correctional Centres, 314 enrolled in the study. The main exclusion from the study was due to prisoners not meeting the inclusion criteria of remaining at the centre for the 16-week duration of the study (n=4,188). Of the remaining 2,412 prisoners, the other main exclusion criteria was the low baseline aggression score on the IBOS (n=1,710, 71%). The baseline and post 16-week intervention mean (95% CI) IBOS score for placebo was 0.678 (0.390, 0.763) and 0.148 (0.027, 0.268) respectively, and for the omega-3 group was 0.580 (0.492, 0.669) and 0.222 (0.101, 0.373), respectively, P(time) = 0.002, P(group) = 0.47, P(interaction) = 0.50. Hence both placebo and omega-3 groups showed improvement on the IBOS scores, with no significant differences between the two groups. Similarly, there were no significant differences between the two groups in any of the secondary measures. In conclusion, omega-3 supplementation did not improve aggressive behaviour in prisoners with low levels of aggressive behaviour, over and above the placebo effect of personal interaction with the prisoners.