Histone deacetylase 3 (HDAC3) has been implicated to play roles in governing cell proliferation. Here we demonstrated that the over-expression of HDAC3 repressed transcription of p15(INK4b) and p21(WAF1/cip1) genes in 293T cells, and that the recruitment of HDAC3 to the promoter regions of these genes was critical to this repression. We also showed that HDAC3 repressed GAL4-Sp1 transcriptional activity, and that Sp1 was co-immunopreciptated with FLAG-tagged HDAC3. We conclude that HDAC3 can repress p15(INK4b) and p21(WAF1/cip1) transcription by interacting with Sp1. Furthermore, knockdown of HDAC3 by RNAi up-regulated the transcriptional expression of p15(INK4b), but not that of p21(WAF1/cip1), implicating the different roles of HDAC3 in repression of p15(INK4b) and p21(WAF1/cip1) transcription. Data from this study indicate that the inhibition of p15(INK4b) and p21(WAF1/cip1) may be one of the mechanisms by which HDAC3 participates in cell cycle regulation and oncogenesis. (c) 2005 Elsevier Inc. All rights reserved.