Despite previous studies suggesting that choline and betaine ameliorate lipid accumulation in rat livers, the relative effectiveness of the two nutrients is unclear. We examined the efficacy of dietary supplementation with choline or betaine in ameliorating lipid accumulation induced by vitamin B₆ (B₆) deficiency in the rat liver. Male Wistar rats were fed control, B₆-deficient, choline-supplemented B₆-deficient, betaine-supplemented B₆-deficient, or both choline and betaine-supplemented B₆-deficient diets (all containing 9 g of l -methionine (Met)/kg) for 35 d. Two experiments were performed, i.e., one using 17 mmol/kg diet choline bitartrate, betaine anhydrous, and the combination and another using 8.5 mmol/kg diet. Rats fed a B₆-deficient diet developed lipid accumulation in the liver with a reduction of plasma lipids induced by the disruption of Met metabolism. However, the addition of 17 mmol/kg diet choline or betaine was sufficient to ameliorate the disruptions of lipid and Met metabolism. Additionally, 8.5 mmol/kg diet choline ameliorated liver lipid deposition, while the same amount of betaine had no significant effects on liver or plasma lipid profiles. Supplementation with choline resulted in a higher liver betaine than that found using the same amount of betaine alone, although the overall liver betaine content was reduced in B₆-deficient rats. Our findings indicate that choline is more effective than betaine in ameliorating B₆ deficiency-related disruptions in Met metabolism and liver lipid accumulation by increasing liver betaine levels.