Two experiments were conducted to investigate the effect of betaine on egg production, lipid metabolism, and osmoregulation in 18-to 42-week-old ISA Brown laying hens. In experiment 1, three hundred and sixty one hens were fed a com-soy basal diet contailing 16% crude protein (CP), 2800 kcal/kg metabolizable energy (ME), 0.33% methionine, and 0, 300, 600, or 1200 mg betaine per kg diet. Egg production, egg weight, feed consumption, feed conversion, and egg quality were measured every eight weeks. Betaine concentration in live and egg were determined along with serum cholesterol, abdominal fat, total serum protein and albumin levels. In experiment 2, twenty thirty-three-week-old laying hens were fed the same diets as those used in experiment 1 in individual cages and the amount of feed and water consumption were measured for two weeks. At the end of experiment 2, all birds were killed to determine blood plasma and ileal osmopressure, arginine vasotocin (AVT), and liver moisture content. In experiment 1, egg production between the treatments during the first eight weeks were not different, whereas the significant increment of egg production were noticed in the birds fed more than 600 ppm betaine after reaching the peak egg production stage (p<0.05). The egg weight was reduced significantly by the betaine supplementation for the first 8 weeks (p<0.05). Feed conversion tended to improve by betaine supplement. Egg quality was not enhanced by betaine supplementation. Liver betaine level increased with betaine feeding compared to the control but betaine concentration in eggs decreased with betaine supplementation. Betaine supplementation elevated the level of serum total cholesterol and triglyeerides compared to the control. Abdominal fat content was increased by betaine supplementation, whereas liver fat content decreased. In experiment 2, water consumption significantly increased in hens fed diets containing 300 and 600 mg betaine/kg (p<0.05) and osmotic pressure of ileal digesta increased with betaine supplement. Liver moisture content was not affected by betaine, but AVT increased in hens fed betaine. The overal results suggested the possibility of using betaine as a feed additives in the laying hens beacuse of its positive contribution to improving egg production and other metabolic parameters related to lipid metabolism.