A novel human coronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV), hascaused outbreaks of a SARS-like illness with high case fatality rate. The reports of its personto-person transmission through close contacts have raised a global concern about itspandemic potential. Here we characterize the six-helix bundle fusion core structure ofMERS-CoV spike protein S2 subunit by X-ray crystallography and biophysical analysis. Wefind that two peptides, HR1P and HR2P, spanning residues 998–1039 in HR1 and 1251–1286 inHR2 domains, respectively, can form a stable six-helix bundle fusion core structure,suggesting that MERS-CoV enters into the host cell mainly through membrane fusionmechanism. HR2P can effectively inhibit MERS-CoV replication and its spike protein-mediatedcell–cell fusion. Introduction of hydrophilic residues into HR2P results in significantimprovement of its stability, solubility and antiviral activity. Therefore, the HR2P analogueshave good potential to be further developed into effective viral fusion inhibitors for treatingMERS-CoV infection.