This study evaluated the effect of a fat blend containing long-chain (LC) n-3 PUFA on bone mineral density (BMD) and bone metabolism in gonad-intact middle-aged male rats (12 months old, n 28). Seven rats were killed on day 0 of dietary intervention to determine the baseline BMD. The remaining rats (seven per group) were fed a diet with one of the following dietary lipid treatments (g/kg diet): 167 g safflower oil + 33 g menhaden oil (N6 + N3 diet, control), 200 g safflower oil (N6 diet, almost devoid of LC n-3 PUFA), or 190 g menhaden oil + 10 g corn oil (N3 diet, rich in LC n-3 PUFA) for 20 weeks. After 20 weeks, all dietary treatment groups had a lower BMD compared with the baseline reference. However, rats fed the N3 diet had the highest bone mineral content and cortical + subcortical BMD compared with those fed the N6 and control N6 + N3 diet. Compared with the control (N6 + N3) group, rats fed the N3 diet had higher values for serum insulin-like growth factor-I, parathyroid hormone, 1,25-(OH)2 vitamin D3 and bone-specific alkaline phosphatase activity, but lower bone NO production and urinary Ca, whereas rats fed the N6 diet had higher bone prostaglandin E2 production and serum pyridinoline. These findings indicate a protective action of LC n-3 PUFA on ageing-induced bone loss in gonad-intact middle-aged male rats through a modulation of local factors and systemic calcitrophic hormones.