One of the most consumed freshwater fish in South America is Nile tilapia. The present study examined the effects of flaxseed oil (FO), source of alfa-linolenic acid (LNA), on the total lipid composition and polyunsaturated fatty acid, n-6 and n-3 PUFA, contents on Nile tilapia (Oreochromis niloticus) liver. Tilapias were given diets with increasing levels 0.00, 1.2, 2.50, 3.75 and 5.00% (w w(-1)) of FO as a replacement of sunflower oil for five months. Fatty acids analysis of methyl esters revealed 45 fatty acids common to all treatments. The increase of flaxseed oil resulted in a decrease in total n-6 PUFA (35.1 to 21.1%) and an increase in n-3 PUFA (3.3 to 18.5%). The diet with LNA underwent sequential desaturation and elongation in liver, leading to an increase in all n-3 PUFA and a decrease in n-6/n-3 ratios (10.7 to 1.1). The manipulation of fatty acids with FO may be used to increase n-3 PUFA and to help balance n-6/n-3 PUFA in dietary supplements, thus, the liver tilapia becomes one product with major nutritional value.