A study was conducted to evaluate the efficiency of guanidinoacetic acid (GAA) in diets containing varying levels of corn replacement with low-tannin sorghum (LTS) for laying hens in the later stage of production. In a 12-wk study, a total of 288 laying hens at 52 wk of age were divided into 6 treatment groups. Each treatment group had 8 replicates, each of which consisted of 6 hens. A 2 × 3 factorial design was used to investigate the impact of substituting corn with LTS at 3 levels (100% corn, 50% LTS, and 100% LTS) with 2 doses of GAA supplementation (0 and 0.6 g/kg). The results indicate that there were interaction effects (P < 0.05) between diet type and GAA supplementation on protein digestibility and AMEn, with the GAA supplement being more effective in the 100% LTS group. Replacing corn with LTS at both levels had no negative effects on performance and metabolic profile. In contrast, the 100% LTS diet increased monounsaturated fatty acids in the yolk (P < 0.05), but decreased the yolk color index, the ratio of polyunsaturated fatty acids (PUFA) to saturated fatty acids (SFA) in the yolk, ileal digestibility of energy, and AMEn when compared to the 100% corn diet (P < 0.05). Regardless of the diet, dietary supplementation with GAA resulted in increases (P < 0.05) in shell-breaking strength, the PUFA to SFA ratio in egg yolk, and concentrations of creatine and nitric oxide in serum. There was also a decrease (P < 0.05) in serum malondialdehyde concentration with GAA supplementation. In conclusion, the positive effects of GAA on protein digestibility and AMEn were found to be more pronounced when corn was completely replaced with LTS. However, the positive effects of GAA on egg-laying performance, eggshell quality, antioxidant status, and yolk fatty acid composition remained consistent regardless of the extent to which corn was substituted with LTS.