Notch1 is a multifunctional transmembrane receptor that regulates cellular differentiation, development, proliferation, and survival in a variety of contexts. We have previously shown that Notch1 may function as a tumor suppressor and that histone deacetylase (HDAC) inhibitors can induce Notch1 expression in some endocrine cancers. Here, we showed that although there was minimal Notch1 expression in follicular thyroid cancer FTC236 and papillary thyroid cancer DRO cells, trans-fection of constitutive Notch1 plasmid into these cells led to growth inhibition, down-regulation of cyclin D1, and up-regulation of p21.Treatment of FTC236 cells with HDAC inhibitors valproic acid (1-4 mmol/L) or suberoyl bishydroxamic acid (10-30 μmol/L) induced functional Notch1 protein expression and suppressed cell growth in a dose-dependent manner.Notch1 siRNA interference blocked the antiproliferative effect of HDAC inhibitors. Western blot analysis revealed the reduction of cyclin D1 and the increase of p21 in HDAC inhibitor-treated cells. These results indicate that HDAC inhibitors activate Notch1 signaling in thyroid cancer cells and lead to the suppression of proliferation by cell cycle arrest. Our findings provide the first documentation of the role of Notch1 signaling as a tumor suppressor in DRO and FTC236 cells, suggesting that Notch1 activation may be a potential therapeutic target for papillary and follicular thyroid cancers. Copyright © 2009 American Association for Cancer Research.