The objective was to elucidate the effects of dietary supplementation with guanidinoacetic acid (GAA) on performance, egg quality and the liver antioxidant activity of laying hens. A total of 128, 72-week-old ISA Brown laying hens were randomly divided into four equal groups (32 birds), and each subgroup had eight replicates (four birds/cage). The control group (GAA0) fed the basal diet with no supplements, while the other experimental groups fed the basal diets supplemented with 0.5 (GAA1), 1.0 (GAA2) and 1.5 (GAA3) g of GAA/kg diet. The experiment lasted for 6 weeks. The addition of GAA at a rate of 1.5 g kg−1 significantly increased the hen-day egg production and egg mass as compared to the control group (P = 0.016 and 0.003, respectively). Although the egg weight was not affected (P = 0.521) by the dietary supplements, the shell ratio, shell thickness, yolk index and Haugh units increased linearly with the increase in the dietary supplements of the GAA (P = 0.036, 0.001, 0.012 and 0.004, respectively). The liver MDA levels decreased linearly with the increment in the dietary levels of the GAA (P = 0.012). Birds in the GAA2 and GAA3 showed a significantly higher liver nitric oxide level (52.50 and 54.21 mg/g, respectively) when compared with GAA0 and GAA1 groups (P = 0.029). Compared to the GAA0 group, all GAA-supplemented groups showed significantly higher liver ATP levels (P = 0.047). In conclusion, the dietary GA supplements at doses of 1.0 or 1.5 g kg−1 may improve the laying performance, antioxidant activity and the status of cellular energy metabolism in laying hens.