Curcumin (Cur), a well-known dietary pigment derived from Curcuma longa, is a promising anticancer drug, but its in vivo target molecules remain to be clarified. Here we report that exposure of human hepatoma cells to Cur led to a significant decrease of histone acetylation. Histone acetyltransferase (HAT) and histone deacetylase (HDAC) are the enzymes controlling the state of histone acetylation in vivo. Cur treatment resulted in a comparable inhibition of histone acetylation in the absence or presence of trichostatin A (the specific HDAC inhibitor), and showed no effect on the in vitro activity of HDAC. In contrast, the domain negative of p300 (a most potent HAT protein) could block the inhibition of Cur on histone acetylation; and the Cur treatment significantly inhibited the HAT activity both in vivo and in vitro. Thus, it is HAT, but not HDAC that is involved in Cur-induced histone hypoacetylation. At the same time, exposure of cells to low or high concentrations of Cur diminished or enhanced the ROS generation, respectively. And the promotion of ROS was obviously involved in Cur-induced histone hypoacetylation, since Cur-caused histone acetylation and HAT activity decrease could be markedly diminished by the antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) or their combination, but not by their heat-inactivated forms. The data presented here prove that HAT is one of the in vivo target molecules of Cur; through inhibiting its activity, Cur induces histone hypoacetylation in vivo, where the ROS generation plays an important role. Considering the critical roles of histone acetylation in eukaryotic gene transcription and the involvement of histone hypoacetylation in the lose of cell viability caused by high concentrations of Cur, these results open a new door for us to further understand the molecular mechanism involved in the in vivo function of Cur.