Given the pivotal role of angiotensin-converting enzyme 2 (ACE2) in mediating viral entry and the genetic divergence observed in ACE2 orthologs across different species, we aimed to elucidate further the molecular intricacies underlying the interactions between severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike (S) protein and ACE2. In this study, we examined the amino acid residues in ACE2 orthologs interacting with SARS-CoV-2 spike receptor-binding domain to identify those with discernible effects on viral binding and entry. Through <i>in vitro</i> mutagenesis and <i>in silico</i> modeling studies of ACE2 variants, we have pinpointed the amino acid substitutions in human ACE2 that affect SARS-CoV-2 binding and entry. This work can significantly advance our understanding of the molecular mechanisms of SARS-CoV-2-host interactions, receptor recognition, viral entry process, and potential therapeutic options targeting coronavirus entry.