<p><strong>Summary:</strong> This study aimed to determine the effect of fish oil on bone mineral density (BMD). There were no differences in the 2-year BMD measures between high and low dose groups after adjusting for baseline BMD. This randomized controlled trial did not demonstrate any efficacy of omega-3 fatty acids on bone loss in adults.</p> <p><strong>Introduction:</strong> The purpose of this study is to investigate whether supplementation with high dose omega-3 fish oil could have an impact on BMD.</p> <p><strong>Methods:</strong> In a multicentre, double-blind randomized controlled trial (RCT) (ACTRN 12607000415404), 202 Australian participants aged ≥40 with knee osteoarthritis (mean age, 61.0 ± 10.0 years; 49 % female) were randomized to receive either high dose (4.5 g eicosapentaenoic acid and docosahexaenoic acid daily) or low dose (0.45 g/day) omega-3 fish oil for 2 years. BMD was assessed at baseline and 2 years by dual energy X-ray absorptiometry.</p> <p><strong>Results:</strong> In subjects with baseline and 2-year assessments, mean standardized BMD at baseline for low or high dose group was 1198 ± 198 and 1157 ± 169 mg/cm<sup>2</sup>, respectively, for the lumbar spine and was 1035 ± 165 and 1017 ± 174 mg/cm<sup>2</sup>, respectively, for the femoral neck. There were no differences in the 2-year BMD measures between high and low dose groups after adjusting for baseline BMD in the complete case regression analyses (lumbar spine 3.7, 95 % confidence interval (CI) −7.9 to 15.3 mg/cm<sup>2</sup> and femoral neck −5.5, 95 % CI −14.9 to 3.9 mg/cm<sup>2</sup>). The findings did not change with additional adjustments of age, gender, study centre and uses of bone-related drugs during the study period as well as using the intention-to-treat analysis or limiting to older participants (≥55 years at the baseline) (all <em>P</em> ≥ 0.25). Mild adverse events such as headache and gastrointestinal intolerance were common but did not occur more frequently in either group. There were no serious adverse events related to the intervention.</p> <p><strong>Conclusion:</strong> A 2-year supplementation with high-dose omega-3 fish oil did not alter bone loss among men and women with knee osteoarthritis.</p>