To find out whether the mutations of HIV-1 Env have influence on the assembly of pseudovirus and their abilities to infect cells, site-directed mutation (A457D)was performed using cycling mutagenesis and selection of mutants with DpnI. Transformation and plasmid purification technologies were used to obtain mutated env clone. Then both the prototype and the mutant were co-transfected with pSG3(delta(env)) to 293FT cells, respectively. Single-cycle infection assay was employed to analyze the effect of the prototype and the mutant on the ability of functional pseudovirus assembly. The transient expression of both the prototype S12-42-1 and mutant S12-42M were confirmed by Western blot essay. The S/CO value was less than 1 for S12-42-1 and 6.65 for S12-42M, demonstrating the functional pseudovirus was generated only for S12-42M. So mutation on HIV-1 Env has influence on the assembly of pseudovirus and their abilities to infect cells.