Severe acute respiratory syndrome (SARS) is an emerging infectious disease caused by a novel coronavirus (SARS-CoV). The binding of SARS-CoV spike (S) protein to cellular angiotensin-converting enzyme 2 (ACE2) is the first step in SARS-CoV infection. Therefore, we assayed the inhibitory effects of small peptides derived from S protein on the binding of S protein to ACE2 and on the S-protein-pseudotyped retrovirus infectivity. SP-4 (residues 192–203), SP-8 (residues 483–494), and SP-10 (residues 668–679) significantly blocked the interaction between S protein and ACE2 by biotinylated enzyme-linked immunosorbent assay, with IC 50 values of 4.30 ± 2.18, 6.99 ± 0.71, and 1.88 ± 0.52 nmol, respectively. Peptide scanning suggested the region spanning residues 660–683 might act as a receptor-binding domain. SP-10 blocked both binding of the S protein and infectivity of S protein-pseudotyped retrovirus to Vero E6 cells. In conclusion, this is the first report of small peptides designed to disrupt the binding of SARS-CoV S protein to ACE2. Our findings suggest that SP-10 may be developed as an anti-SARS-CoV agent for the treatment of SARS-CoV infection.