Abstract: ThVHAc1 gene was reported to be responsive to drought, salt and heavy metal stresses, and overexpressed ThVHAc1 yeast could improve effectively multi-stress tolerance. To further understand the stress regulation mechanism of ThVHAc1, we screened the upstream regulators that could specifically recognize the cis-elements DOF and MYB in the ThVHAc1 promoter by yeast one-hybrid assay. The results showed that the ThMYB3 gene could specifically recognize the MYB motif and the promoter segment that contains MYB motif, and the ThDof2 gene could specifically recognize the DOF motif and the promoter segment that includes DOF motif in the ThVHAc1 promoter. However, ThMYB3 and ThDof2 could not recognize the mutated motifs and promoter segments containing these mutated motifs. Then ThMYB3 and ThDof2 were independently constructed as effecter vector, and MYB and DOF motifs as well as their mutates and promoters were independently constructed as reporter vectors for co-expression assays. And the results of co-transient expression of the effecters and reporters confirmed that ThDof2 and ThMYB3 can specifically bind to the ThVHAc1 promoter. ThMYB3 and reporters containing MYB motif and ThVHAc1 promoter segments including MYB motif, ThDof2 and reporters containing DOF motif and the ThVHAc1 promoter segments including DOF motif could stain the GUS expression in tobacco leaves and showed high GUS activities. However, other co-expression referred to mutated motifs could not activate the GUS expression, indicating that only normal cis-elements and promoter segments could be recognized by effecters to activate the GUS expression. Our study suggests the effectiveness of ThMYB3 and ThDof2 as ThVHAc1 upstream regulators.