The n-6/n-3 ratio of polyunsaturated fatty acids (PUFAs) in enteral feeding formulas is not considered to be important, because the short-term administration of these formulas has been the norm for postoperative digestive organs. However, the long-term administration of enteral feeding formulas has been increasingly recommended for patients with aging-associated aphagia. This study is aimed at investigating the effect of the long-term administration of an enteral feeding formula containing n-3 polyunsaturated fatty acids (PUFAs) on the fatty acid composition of endogenous phospholipids. Rats, which were initially fed a diet lacking n-3 PUFAs for 2 generations, were subsequently fed an enteral feeding formula containing or lacking n-3 PUFAs for 12 weeks (n = 10). Then, the fatty acid composition of phospholipids in the brain, liver, red blood cells, and plasma of the rats was analyzed. Although the fatty acid composition of neural tissues is suggested to be not affected by diet, the n-6/n-3 ratio of phospholipids in the brains of rats that were fed an enteral feeding formula containing n-3 PUFAs was significantly lower than those of rats that were fed a formula lacking n-3 PUFAs. The enteral feeding formula containing n-3 PUFAs may be effective for the regulation of brain functions and the prevention of atherosclerosis.