Cancer cells have complex, unique characteristics that distinguish them from normal cells, such as increased growth rates and evasion of anti-proliferative signals. Global inhibition of class I and II histone deacetylases (HDACs) stops cancer cell proliferation in vitro and has proven effective against cancer in clinical trials, at least in part, through transcriptional reactivation of the p21 WAF1/Cip1 gene. The HDACs that regulate p21 WAF1/Cip1 are not fully identified. Using small interfering RNAs, we found that HDAC4 participates in the repression of p21 WAF1/Cip1 through Sp1/Sp3-, but not p53-binding sites. HDAC4 interacts with Sp1, binds and reduces histone H3 acetylation at the Sp1/Sp3 binding site-rich p21 WAF1/Cip1 proximal promoter, suggesting a key role for Sp1 in HDAC4-mediated repression of p21 WAF1/Cip1. Induction of p21 WAF1/Cip1 mediated by silencing of HDAC4 arrested cancer cell growth in vitro and inhibited tumor growth in an in vivo human glioblastoma model. Thus, HDAC4 could be a useful target for new anti-cancer therapies based on selective inhibition of specific HDACs.