The N121 site on the spike protein of SARS‐CoV‐2 is associated with heme and its metabolite, biliverdin, which can affect antibody binding. Both N121T and N121S substitutions have been observed in natural conditions and in a hamster model of dual infection with SARS‐CoV‐2 and Influenza A virus. Serum pseudotype neutralization assays against HIV‐1 particles carrying wild‐type, N121T, and N121S spikes with immune mouse and human sera revealed that N121T and N121S mutations had a greater impact on serum neutralization than biliverdin treatment. Although N121T and N121S substitutions are not currently major SARS‐CoV‐2 variants of concern, this study could provide fundamental information to prepare for potential future mutations at the N121 site of SARS‐CoV‐2.