We previously reported that both sodium butyrate and trichostatin A (TSA), both of which are known as inhibitors of histone deacetylase, arrest human tumor cells at G1 and G2-M and activate the cyclin-dependent kinase inhibitor, the p21/WAF1/Cip1 gene promoter, through the Sp1 sites. In this study, we identified Sp1 and Sp3 as major factors binding to the Sp1 sites of the p21/WAF1/Cip1 promoter in MG63 cells through electrophoretic mobility shift assays and showed that TSA treatment did not change their binding activities. However, GAL4-Sp3 but not GAL4-Sp1 fusion protein supported the TSA-mediated gene induction from a luciferase reporter plasmid driven by five GAL4 DNA-binding sites. Moreover, the ectopic expression of dominant negative Sp3 repressed the enhancement by TSA of the p21/WAF1/Cip1 promoter and Sp1 site-driven promoter. Taken together, these results suggest that histone deacetylase inhibitor up-regulates p21/WAF1/Cip1 transcription by Sp3 but not by Sp1.