Different levels of metabolizable energy (ME) and the inclusion of guanidinoacetic acid (GAA) in the diet of 53-week-old Lohmann LSL-CLASSIC hens were used to evaluate its effect on reproductive parameters, egg quality, intestinal morphology, and the immune response. Six diets were used in a 3 × 2 factorial design, with three levels of ME (2850, 2800, and 2750 kcal/kg), and with (0.08%) or without the inclusion of GAA. The addition of GAA to diets with low levels of ME increased (p < 0.05) egg production and egg mass. Moreover, hens fed with 2800 kcal/g without GAA had the highest concentration (p < 0.05) of serum interleukin IL-2, while those fed diets with the same amount of ME but supplemented with 0.08% GAA had the lowest concentration. Finally, the inclusion of 0.08% GAA increased (p < 0.05) the concentration of vascular endothelial growth factor (VEGF), regardless of the ME level in the diet. This study highlights the potential role of GAA in decreasing the energy level of ME (50–100 kcal/g) in the feeding of hens and in the modulation of specific immune responses. Further research is recommended to fully understand the mechanisms of action of GAA on the mechanism target of rapamycin and its relationship with the immune response.