Synthesis of a series of novel phosphorylated ganciclovir derivatives with bioactive amines/aminoacid esters as substituents was accomplished. These compounds were structurally characterized by IR, NMR (¹H, ¹³C, ³¹P), mass spectra and CHN analysis. The compounds (5a–m) have been evaluated for its in vitro and in vivo immunomodulatory activities. The amino acid ester substituted ganciclovir derivatives, especially the 5g and 5i, increased the intracellular killing activity of the stimulated neutrophils. The in vivo experiment results show that the administration of compounds 5g and 5i (8 mg/kg body weight) to diet-induced immune impaired obese rats ameliorated the significant increase in immune cell counts (lymphocytes, neutrophils and monocytes). In addition, TNF-a, IL-6 and IL-1 secretions were considerably restored to normal by the compounds 5g and 5i with regulation in the release of C-reactive protein suggesting their potentiating effect on immune system dysfunction. These in vitro and in vivo results suggest that the phosphorylated ganciclovir derivatives 5g and 5i are strong immunomodulators.