Guanidinoacetic acid (GAA) can effectively improve the metabolism of energy and proteins by stimulating creatine biosynthesis. We present a study exploring the impact of GAA on production performance, serum biochemistry, meat quality and rumen fermentation in Hu sheep. A total of 144 weaned male Hu sheep (body weight 16.91 ± 3.1 kg) were randomly assigned to four groups with three replicates of twelve sheep in each group. The diets were supplemented with 0 (CON), 500 (GAA-1), 750 (GAA-2) and 1000 mg/kg (GAA-3) of GAA (weight of feed), respectively. After a comprehensive 90-day experimental period, we discovered that the supplementation of GAA had a remarkable impact on various muscle parameters. Specifically, it significantly enhanced the average daily growth (ADG) of the animals and improved the shear force and fiber diameter of the muscle, while also reducing the drip loss and muscle fiber density. Furthermore, the addition of GAA to the feed notably elevated the serum concentrations of high-density lipoprotein cholesterol (HDL-C), total protein (TP) and globulin (GLB), as well as the enzyme activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). Concurrently, there was a decrease in the levels of triglycerides (TG) and malondialdehyde (MDA) in the serum. In addition, GAA decreased the pH and the acetate-to-propionate ratio and increased the total volatile fatty acids (TVFA) and ammoniacal nitrogen (NH3-N) levels of rumen fluid. Additionally, GAA upregulated acetyl-CoA carboxylase (ACC) gene expression in the Hu sheep's muscles. In conclusion, our findings suggest that GAA supplementation not only enhances muscle quality but also positively affects serum biochemistry and ruminal metabolism, making it a potential candidate for improving the overall health and performance of Hu sheep.