Author summary Human cytomegalovirus (HCMV) infection is the foremost viral cause of congenital birth defects. Attempts to vaccinate against HCMV brought moderate, though not sufficient, reduction in infection. Moreover, phase II clinical trial with gB formulated in MF59 adjuvant did not induce a neutralizing antibody response. Here, we identify the antigenic domain 5 (AD5) of gB as the target of most neutralizing antibodies. Moreover, functionalization of AD5 on self-assembling nanoparticles leads to a gB vaccine candidates generating high antibody binding and neutralizing titers. Interestingly, it was recently reported that most antibodies targeting AD5 could inhibit cell fusion, a discovery on interest as HCMV is thought to disseminate within the host primarily through direct cell-to-cell spreading. Our data not only provides new vaccine candidates but may also have important implications for understanding cellular fusion promoted by HCMV. Human cytomegalovirus (HCMV) is the primary viral cause of congenital birth defects and causes significant morbidity and mortality in immune-suppressed transplant recipients. Despite considerable efforts in vaccine development, HCMV infection still represents an unmet clinical need. In recent phase II trials, a MF59-adjuvanted gB vaccine showed only modest efficacy in preventing infection. These findings might be attributed to low level of antibodies (Abs) with a neutralizing activity induced by this vaccine. Here, we analyzed the immunogenicity of each gB antigenic domain (AD) and demonstrated that domain I of gB (AD5) is the main target of HCMV neutralizing antibodies. Furthermore, we designed, characterized and evaluated immunogenic responses to two different nanoparticles displaying a trimeric AD5 antigen. We showed that mice immunization with nanoparticles induces sera neutralization titers up to 100-fold higher compared to those obtained with the gB extracellular domain (gB(ECD)). Collectively, these results illustrate with a medically relevant example the advantages of using a general approach combining antigen discovery, protein engineering and scaffold presentation for modern development of subunit vaccines against complex pathogens.