Refocusing of B-cell responses can be achieved by preserving the overall fold of the antigen structure but selectively mutating the undesired antigenic sites with additional N-linked glycosylation motifs for glycan masking the vaccine antigen. We previously reported that glycan-masking recombinant H5 hemagglutinin (rH5HA) antigens on residues 83, 127, and 138 (g127 + g138 or g83 + g127 + 138 rH5HA) elicited broader neutralizing antibodies and protection against heterologous clades/subclades of high pathogenic avian influenza H5N1 viruses. In this study, we engineered the stably expressing Chinese hamster ovary (CHO) cell clones for producing the glycan-masking g127 + g138 and g83 + g127 + g138 rH5HA antigens. All of these glycan-masking rH5HA antigens produced in stable CHO cell clones were found to be mostly oligomeric structures. Only the immunization with the glycan-masking g127 + g138 but not g83 + g127 + g138 rH5HA antigens elicited more potent neutralizing antibody titers against four out of five heterologous clades/subclades of H5N1 viral strains. The increased neutralizing antibody titers against these heterologous viral strains were correlated with the increased amounts of stem-binding antibodies, only the glycan-masking g127 + g138 rH5HA antigens can translate into more protection against live viral challenges. The stable CHO cell line-produced glycan-masking g127 + g138 rH5HA can be used for H5N1 subunit vaccine development.