Since many connections exist between vitamin B6 and lipid metabolism, we aim to investigate the lipemic effect of different dietary intakes of polyunsaturated fatty acids in rats fed a vitamin B6 deficient diet. Diets were either vitamin B6 deficient (-B6) or vitamin B6 sufficient, pair-fed to the deficient group (PF) and ad libitum (N). The diets were combined with normal lipid (LC: soya bean-coconut-palm oils) and fish oil (FO: soya bean-fish oil). The fish oil diet with sufficient vitamin B6 content caused an increase in n-3 long chain polyunsaturated fatty acids and a decrease in arachidonic acid. In the -B6 group fed a normal lipid diet, the arachidonic acid percentage decreased and the linoleic acid percentage increased; in the -B6 group fed fish oil these changes in fatty acid composition, already consequent upon dietary intake of n-3 long chain polyunsaturated fatty acids, did not show further variations. In the dietary condition of vitamin B6 deficiency, plasma cholesterol content increased in rats fed a lipid control diet, whereas no hypocholesterolemic effect was observed in those fed a fish oil diet. Plasma triglyceride contents were not influenced by dietary lipid quality because, in all conditions, the lower food intake of the PF groups caused a decrease and vitamin B6 deficiency caused an elevation in triglyceride contents which reached those of the ad libitum groups. The study highlights the interaction between vitamin B6 and polyunsaturated fatty acids and the opportunity of dietary intake of fish oil to counterbalance some effects of vitamin B6 deficiency.