Abstract: Objective To construct a HIV-1 gp120 transgenic mice (gp120 Tg) with vimentin (VIM) gene knockout. Methods Female HIV-1 gp120 Tg mice were mated to VIM heterozygote mice (F0). All the offspring mice were derived from these original founders so that both genotypes had the same mixed genetic background. The F1 mice were bred to generate of VIM+/+, VIM-/-, VIM+/+/gp120 Tg and VIM-/-/gp120 Tg mice. PCR was performed for genotyping of the mice, and the expressions of VIM and gp120 in the brain tissues were examined using immunoblotting. Results The results of PCR showed the presence of the target bands in VIM +/+ , VIM-/-, VIM +/+/gp120 Tg and VIM-/-/gp120 Tg mice. In VIM-/-/gp120 Tg mice, gp120 expression was detected throughout the brain regions while no VIM expression was detected. Conclusion We generated gp120 transgenic mouse models with VIM gene knockout, which facilitate the exploration of the role of VIM in gp120-induced neurotoxicity.