The role of NOTCH1 as an oncogene or tumor suppressor appears to be cell type-specific. Medullary thyroid cancer (MTC) cells characteristically express the transcription factor ASCL1 (achaete-scute complex-like 1) as well as high levels of the neuroendocrine (NE) markers calcitonin and chromogranin A (CgA). In this study, we show that the active NOTCH1 intracellular domain is absent in human MTC tumor tissue samples and MTC-TT cells. To determine the effects of NOTCH1 expression, we created a doxycycline-inducible NOTCH1 intracellular domain in MTC cells (TT-NOTCH cells). Treatment of TT-NOTCH cells with doxycycline led to dose-dependent induction of NOTCH1 protein with corresponding decreases in ASCL1 protein and NE hormones. ASCL1 promoter-reporter assay and Northern analysis revealed that ASCL1 reduction by NOTCH1 activation is predominantly via silencing of ASCL1 gene transcription. Overexpression of ASCL1 in MTC cells indicated that CgA expression is highly dependent on the levels of ASCL1. This was further confirmed by experiments using small interfering RNA against ASCL1, in which reduction in ASCL1 led to reduction in both CgA and calcitonin. Furthermore, we demonstrate that NOTCH1 signaling activation leads to ERK1/2 phosphorylation, but that reduction in NE markers is independent of ERK1/2 activation. Activation of NOTCH1 resulted in significant MTC cell growth inhibition. Notably, reduction in MTC cell growth was dependent on the level of NOTCH1 protein present. Moreover, no increase in growth upon expression of ASCL1 in NOTCH1-activated cells was observed, indicating that the growth suppression observed upon NOTCH1 activation is independent of ASCL1 reduction. Mechanistically, we show that MTC cell growth inhibition by NOTCH1 is mediated by cell cycle arrest associated with up-regulation of p21.