Vimentin, a member of the intermediate filament protein family, is regulated both developmentally and tissue specifically. It is also a marker of the metastatic potential of many tumor cells. Pre viously, the human vimentin promoter has been shown to contain multiple elements for the binding of both positive‐ and negative‐acting regulatory factors. Transient transfection analysis of various vimentin 5′‐end promoter sequences and mutants thereof fused to a reporter gene further defined two regulatory elements, a positive element that binds Sp1 and a negative element that binds the protein ZBP‐89. ZBP‐89 has been shown to be either a repressor or an activator of gene expression, depending on the promoter. Here, we show that for vimentin, both ZBP‐89 and ZBP‐99 repress reporter gene expression in Schneider (S2) cells. Deletion constructs confirm that the glutamine‐rich region of Sp1 is required to enhance vimentin transcription, whereas the N‐terminus of ZBP‐89 is required to interact with Sp1 and repress gene expression. The overexpression of hTAF_II130 can alleviate ZBP‐89 repression in S2 cells, suggesting how ZBP‐89 might serve to block gene expression.