Suberoylanilide hydroxamic acid (1), as well as other histone deacetylase (HDAC) inhibitors, are promising, targeted anticancer agents. Curcumin (2), a possible antitumor agent, exhibits a HDAC inhibiting effect but with a different mechanism, and was proposed to synergize with other drugs, including HDAC inhibitors. The present study was undertaken to evaluate the possible inhibitory effects of 1 and 2 combinations on the growth of nine human cancer cell lines. Drug combinations resulted in an antagonistic cytotoxic effect, as characterized by the Loewe additivity model, observed in all the cell lines. On the other hand, histone hyperacetylation was synergistically or at least additively induced by 1 and 2 combinations, in four cell lines tested. Despite the enhanced histone acetylation, 1 plus 2 produced a significant antagonism in the induced activation of downstream p21(CIP/WAF1) expression. Concomitantly, induced reactive oxygen species (ROS) production was antagonistically diminished in combinations especially at low concentration of 2. We conclude that 1 and 2 exert an antagonistic cytotoxicity on a variety of cancer cell lines, and suggest that mechanisms mediating their antagonism lie at levels of p21(CIP/WAF1) expression and ROS production, rather than at histone acetylation.