To further investigate on the structure-activity relationships of immunosuppressive Astin C, seventeen analogues 1-17 were designed and synthetized via amino acid substitution strategy by the solid-phase peptide synthesis method for the first time. In comparison with Astin C (IC50 = 12.6 ± 3.3 μM), only compounds 2 (IC50 = 38.4 ± 16.2 μM), 4 (IC50 = 51.8 ± 12.7 μM), 5 (IC50 = 65.2 ± 15.6 μM), and 8 (IC50 = 61.8 ± 12.4 μM) exhibited immunosuppressive activity in the Lymph node cells of mice. These results showed that the Astin C analogues containing D-amino acid residues, hydrophobic long-chain alkyl substituents, and aryl substituents performed better than those carrying hydrophilic amino acid residues and short-chain alkyl substituents. Moreover compounds 15, 16, and 17 had no immunosuppressive activity, which suggested that cis-3,4-dichlorinated proline played an important role in the immunosuppressive activity of Astin C.