Maternal vitamin B12 deficiency leads to an adverse pregnancy outcome and increases the risk for developing diabetes and metabolic syndrome in mothers in later life. Our earlier studies have demonstrated that vitamin B12 and n-3 polyunsaturated fatty acids (PUFA) are interlinked in the one carbon cycle. The present study for the first time examines the effect of maternal n-3 PUFA supplementation to vitamin B12 deficient or supplemented diets on pregnancy outcome, fatty-acid status and metabolic variables in Wistar rats. Pregnant dams were assigned to one of the following groups: control, vitamin B12 deficient, vitamin B12 supplemented, vitamin B12 deficient + n-3 PUFA or vitamin B12 supplemented + n-3 PUFA. The amount of vitamin B12 in the supplemented group was 0.50 μg kg(-1) diet and n-3 PUFA was alpha linolenic acid (ALA) 1.68, eicosapentaenoic acid 5.64, docosahexaenoic acid (DHA) 3.15 (g per 100g fatty acids per kg diet). Our findings indicate that maternal vitamin B12 supplementation did not affect the weight gain of dams during pregnancy but reduced litter size and weight and was ameliorated by n-3 PUFA supplementation. Vitamin B12 deficiency or supplementation resulted in a low percentage distribution of plasma arachidonic acid and DHA. n-3 PUFA supplementation to these diets improved the fatty-acid status. Vitamin B12 deficiency resulted in higher homocysteine and insulin levels, which were normalised by supplementation with either vitamin B12 or n-3 PUFA. Our study suggests that maternal vitamin B12 status is critical in determining pregnancy outcome and metabolic variables in dams and that supplementation with n-3 PUFA is beneficial.