In a previous work, taking advantage of the gene-array screening technology, we analysed the effects of histone deacetylase (HDAC) inhibitor sodium butyrate (NaBt), on gene transcription in HT29 human adenocarcinoma cell line. In this study, we focused our attention on p55CDC/Cdc20 gene, whose expression was dramatically reduced by NaBt treatment. Mammalian p55CDC/Cdc20 interacts with the anaphase promoting complex/cyclosome (APC/C), and is involved in regulating anaphase onset and late mitotic events. Using NaBt and trichostatin A (TSA), a member of the HDAC inhibitor family, we showed that both HDAC inhibitors totally downregulated p55CDC/Cdc20 transcription and expression. Cell cycle analysis demonstrated that NaBt arrested HT29 cells in G0/G1 phase, while TSA caused a double block in G0/G1 and G2/M phases. Moreover, p55CDC/Cdc20 showed maximal expression in S and G2/M phases of HT29 cell division cycle. Based on this evidence, and by means of specific cell cycle modulators, such as nocodazole and hydroxyurea, we demonstrated that both TSA and NaBt were responsible for loss of p55CDC/Cdc20 expression, but with different mechanisms of action. Taken together, these results suggest that targeting molecules involved in spindle mitotic checkpoint, such as p55CDC/Cdc20, might account for the high cytotoxicity of HDAC inhibitors versus malignant cells.