Objective To study the influence of the modification of the special neutralizing epitopes of the HIV-1 envelope (Env) on its assembly of functional pseudovirus and neutralizing activity. Methods Site-directed mutations were performed using cycling mutagenesis and selection of mutants with Dpn I . With this method, the 2G12 and 2F5 neutralizing epitopes were integrated into Env of subtype BC which was without the two epitopes, then the capability of forming pseudovirus and the neutralizing activity against 2G12 and 2F5 were compared with pre-modified Env. Results The special Env neutralizing epitopes of five HIV pseudovirus (BC02, BC03, BC04, BC05 and BC12) were modified. Among the five pseudovirus, BC04 and BC12 pseudovirus can't be formed after the 2G12 epitope was modified, whilst the BC02, BC03 and BC05 pseudovirus can be formed after the 2G12 and 2F5 epitopes were added, and there was no variation of the pseudovirus titer; On the aspect of neutralizing activity, BG03 pseudovirus against 2G12 and 2F5 was enhanced, BC02 and BC05 pseudovirus against 2F5 was enhanced while which against 2G12 was not changed. Conclusion The modification of 2G12 epitope influences the forming of pseudovirus and the addition of neutralizing epitopes can enhance the neutralizing activity of pseudovirus, which offers new approach for the optimization of HIV immunogen.