Objective To construct eukaryotic expression vectors of vacuolar protein sorting 4B (VPS4B) gene with site-directed mutagenesis. Methods VPS4B gene was amplified by RT-PCR from Huh7 cells and cloned into the eukaryotic vector pXF3H. Site-directed mutagenesis method based on overlap extension PCR was used to construct two dominant negative (DN) mutants, K180Q and E235Q. The point mutation was verified by DNA sequencing. The mammalian hepatoma cell HepG2 was transfected with VPS4B and the 2 mutants. The expression of the fused proteins was determined by Western blot. Results VPS4B gene was cloned and the eukaryotic vector pXF3H-VPS4B was constructed. The mutants were obtained by overlap extension PCR. DNA sequencing showed that AAG at 538540 bp site, which encoded the 180amino acid, was changed to CAG, and in another mutant site, GAA at 703705 bp site, which encoded the 235amino acid, was changed to CAA. No mutation in other base pair sites of the recombinant plasmids. The fusion protein HAVPS4B was detectable by Western blot in HepG2 cells transfected with the 2 mutant vectors. Conclusion The eukaryotic expression vectors for VPS4B and its two DN mutants K180Q and E235Q were successfully implemented.