In this study, the effects of different levels of dietary guanidinoacetic acid (GAA) supplementation (0, 0.6, 1.2 and 1.8 g/kg) in laying quails was evaluated in a completely randomised design comprised of four dietary treatments of six replicates each, from 56 to 154 d of age. The highest laying rate was obtained with 1.8 g/kg of GAA, but the optimum egg weight, egg mass, shell and yolk weight were observed at 1.2 g/kg of GAA. The villus height and villus height: crypt depth ratio of ileum and apparent ileal digestibility of dry matter, crude protein, ether extract and ash were linearly and quadratically affected by GAA supplementation. Maximum glutathione peroxidase and superoxide dismutase enzyme activity and total antioxidant capacity, but minimum malondialdehyde in the serum were observed at 1.2 g/kg of GAA. The highest concentrations of very low-density lipoprotein-cholesterol, total cholesterol and creatine (CRE) were observed at 1.2 g/kg of GAA, whereas the serum low-density lipoprotein-cholesterol (LDL) was at its lowest level. The CRE, nitric oxide and homocysteine concentrations were highest when GAA at 1.8 g/kg was fed, but the highest concentration of total protein and VLDL were observed at 1.2 g/kg of GAA. The betaine content of liver was at its lowest level at 1.8 g/kg GAA supplementation, but the highest levels of total protein and total lipids were observed at 0.6 and 1.8 g/kg of GAA. In conclusion, the results indicate that GAA, especially at 1.2 g/kg, could improve performance, antioxidant status and intestinal performance in laying quails. Highlights Supplementation of GAA up to 1.8 g/kg of the laying quail diet increased egg production rate but liver betaine content decreased by inclusion of more than 1.2 g/kg of GAA. Supplementation of GAA at all studied levels improved nutrient digestibility while the benefits of GAA on intestinal morphology observed at inclusion levels of 0.6 and 1.2 g/kg. The serum and liver antioxidant status of laying quails improved by dietary addition of GAA at 0.6 and 1.2 g/kg levels.