Here we report a hydroacid named DWP0016, which exhibited HDAC inhibition and induced p53 acetylation in U251 glioblastoma cells. DWP0016 effectively inhibited the cell growth of U251 cells and other four carcinoma cell lines but did not affect the normal cells. Cell cycle distribution analysis showed DWP0016 arrested at G1 phase cell cycle dose-dependently in U251 cells. DWP0016 induced caspase-dependent and independent apoptosis in U251 cells, which was identified by flow cytometry analysis, caspases activity analysis, Western blotting assay, and caspases inhibition. Mechanisms research suggested that DWP0016 activated transcription and acetylation of tumor suppressor p53. DWP0016 regulated p300, CBP, and PCAF to facilitate p53 acetylation at lys382 in U251 cells. In addition, activation of p53 by DWP0016 promoted PUMA to catalyze mitochondrial pathway. Besides, siRNA assay indicated p53 was the key gene to induce growth inhibition, cell cycle arrest, and apoptosis in DWP0016 treated U251 cells. Conclusively, our results show DWP0016 is a potent HDAC inhibitor and the anti-tumor activity is consistent with its intended p53 activation mechanisms. These findings indicate the promising antitumor potential of DWP0016 for further glioblastoma treatment applications. J. Cell. Biochem. 114: 1498–1509, 2013. © 2013 Wiley Periodicals, Inc.