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This study aimed to investigate the effects of in ovo feeding (IOF) of L-arginine (Arg) on energy metabolism in post-hatch broilers. A total of 720 eggs was randomly assigned to 3 treatments: 1) non-injected control group, 2) 0.75% NaCl diluent-injected control group, and 3) 1.0% Arg solution-injected group. At 17.5 d of incubation, 0.6 mL of each solution was injected into the amniotic fluid of each egg of injected groups. After hatching, 80 male chicks were randomly assigned to each treatment group with 8 replicates per group. The results showed that IOF of Arg increased glycogen and glucose concentrations in the liver and pectoral muscle of broilers at hatch (P < 0.05). The plasma glucose and insulin levels were higher in the Arg group than in the non-injected and diluent-injected control groups (P < 0.05). Meanwhile, IOF of Arg enhanced the hepatic glucose-6-phosphatase (G6P) activity at hatch (P < 0.05). There was no difference in hexokinase (HK) or phosphofructokinase (PFK) enzyme activities in the pectoral muscle in all groups. Further, IOF of Arg increased the phosphoenolpyruvate carboxykinase (PEPCK) and fructose-1,6-bisphosphatase (FBP) mRNA expressions at hatch (P < 0.05). In addition, broilers in the Arg group had a higher mRNA expression of glycogen synthase and a lower expression of glycogen phosphorylase in the liver and pectoral muscles than in the non-injected controls at hatch (P < 0.05). In conclusion, IOF of Arg solution enhanced liver and pectoral muscle energy reserves at hatch, which might be considered as an effective strategy for regulating early energy metabolism in broilers.