DNA methylation and chromatin structure are two modes of epigenetic control of genome function. Although it is now well established that chromatin silencing could lead to DNA methylation, the relation between chromatin activation and DNA demethylation is unclear. It was generally believed that expression of methylated genes could only be restored by demethylating agents, such as 5-aza-deoxycytidine (5-azaCdR), and that inhibition of histone deacetylation by Trichostatin A (TSA) only activates transcription of unmethylated genes. In this report, we show that increase of histone acetylation by TSA was associated with a significant decrease in global methylation. This global demethylation occurs even when DNA replication is blocked by hydroxyurea, supporting a replication-independent-mechanism of demethylation. TSA also induces histone acetylation, demethylation and expression of the methylated E-CADHERIN and RARβ2 genes. However, the genome-wide demethylation induced by TSA does not affect all methylated tumor suppressor genes equally suggesting that induction of acetylation and demethylation by TSA shows some gene selectivity. Taken together, our data provide evidence for a reversible crosstalk between histone acetylation and DNA demethylation, which has significant implications on the use of HDAC inhibitors as therapeutic agents.