Recombinant proteins are essential in the development of subunit vaccines. In the design of many recombinant proteins, polyhistidine residues are added to the N- or C-termini of target sequences to facilitate purification. However, whether the addition of tag residues influences the immunogenicity of proteins remains unknown. In this study, the tag-free SARS-CoV-2 RBD and His-tag SARS-CoV-2 RBD proteins were investigated to determine whether there were any differences in their receptor binding affinity and immunogenicity. The results showed that the tag-free RBD protein had a higher affinity for binding with hACE2 receptors than His-tag RBD proteins (EC: 1.78 µM vs. 7.51 µM). On day 21 after primary immunization with the proteins, the serum ELISA titers of immunized mice were measured and found to be 1:1418 for those immunized with tag-free RBD and only 1:2.4 for His-tag RBD. Two weeks after the booster dose, tag-free-RBD-immunized mice demonstrated a significantly higher neutralizing titer of 1:369 compared with 1:7.9 for His-tag-RBD-immunized mice. Furthermore, neutralizing antibodies induced by tag-free RBD persisted for up to 5 months and demonstrated greater cross-neutralization of the SARS-CoV-2 Delta variant. Evidence from Western blotting showed that the serum of His-tag-RBD-immunized mice recognized irrelevant His-tag proteins. Collectively, we conclude that the addition of a polyhistidine tag on a recombinant protein, when used as a COVID-19 vaccine antigen, may significantly impair protein immunogenicity against SARS-CoV-2. Antibody responses induced were clearly more rapid and robust for the tag-free SARS-CoV-2 RBD than the His-tag SARS-CoV-2 RBD. These findings provide important information for the design of antigens used in the development of COVID-19 subunit vaccines.