question: Bats carry diverse severe acute respiratory syndrome-related coronaviruses (SARSr-CoVs). The possible interspecies transmission of SARSr-CoVs from bats to humans has caused two severe CoV pandemics, the SARS pandemic in 2003 and the recent COVID-19 pandemic. The receptor utilization of SARSr-CoV plays the key role in viral infection in determining the host range and the interspecies transmission ability of the virus. Both the SARS-CoV and SARS-CoV-2 use angiotensin-converting enzyme 2 (ACE2) as their receptor. Previous studies showed that WIV1 strain, the first living coronavirus isolated from bat using ACE2 as its receptor, is the prototype of SARS-CoV. The receptor-binding domain (RBD) in the spike protein (S) of SARS-CoV and WIV1 is responsible for ACE2 binding and medicates the viral entry. Comparing to human SARS-CoV, the bat WIV1 has three distinct amino acid residues (442, 472, and 487) in RBD. This study aimed at exploring whether these three residues could alter the receptor utilization of SARSr-CoVs. We replaced the three residues in SARS-CoV (BJ01 strain) S with their counterparts in WIV1 S, and then evaluated the change of their utilization of bat, civet, and human ACE2s using a lentiviral pseudovirus infection system. To further validate the S-ACE2 interactions, the binding affinity between the RBDs of these S proteins and different ACE2s were verified by flow cytometry. The results showed that the single amino acid substitution Y442S in the RBD of the SARS-CoV BJ01 spike protein enhanced its utilization of bat ACE2 and its binding affinity to bat ACE2. On the contrary, the reverse substitution in WIV1 S (S442Y) significantly attenuated the pseudovirus utilization of bat, civet and human ACE2s for cell entry, and the binding affinity was also decreased notably. These results suggest that the S442 is critical for WIV1 adapting in bats as its natural host. These findings will enhance our understanding of host adaptations and cross-species infections of coronaviruses, contributing to the prediction and prevention of coronavirus epidemics.