BACKGROUND: Betaine is a derivative of three methylates of glycine amino acids, found in the body of many animals. Objectives: The study was conducted to investigate the effects of betaine supplementation in the diet on gene expression and activities of lipogenic enzymes, and lipid levels of blood and liver in broilers. Methods: A total of 320 broilers were evaluated (Ross 308) in a completely randomized design with 4 treatments and 4 replicates, with the diets consisted of the control treatment which did not use the betaine supplementation. The second, third and fourth treatments contained 0.05, 0.08 and 0.11% betaine hydrochloride 98%, respectively. Results: Betaine supplementation to the diet has no significant effect on lipoprotein lipase gene expression, and the activities of enzyme Acetyl-CoA carboxylase and fatty acid synthase (p> 0.05). Betaine supplementation caused a significant decrease in liver cholesterol and triglyceride chicks fourth treatment (containing betaine hydrochloride 0.11 percent) compared to the control group (p<0.05). Conclusions: In general, the results show that in broilers lipoprotein lipase gene expression and the activities of enzyme Acetyl-CoA carboxylase and fatty acid synthase are less influenced by betaine.