The cell fate determinant Numb influences developmental decisions by antagonizing the Notch signaling pathway. However, the underlying molecular mechanism of this inhibition is poorly understood. Here we report that the mammalian Numb protein promotes the ubiquitination of membrane-bound Notch1 receptor. Furthermore, Numb expression resulted in the degradation of the Notch intracellular domain following activation, which correlated with a loss of Notch-dependent transcriptional activation of the Hes1 promoter as measured by a Hes1 luciferase reporter assay. The phosphotyrosine-binding (PTB) domain of Numb was required for both Notch1 ubiquitination and down-regulation of Notch1 nuclear activity. Numb-mediated ubiquitination of Notch1 was not dependent on the PEST region, which was previously shown to mediate Sel10-dependent ubiquitination of Notch in the nucleus, suggesting a distinct E3 ubiquitin ligase is involved. In agreement we demonstrate that Numb interacts with the cytosolic HECT domain-containing E3 ligase Itch and that Numb and Itch act cooperatively to promote ubiquitination of membrane-tethered Notch1. These results suggest that Numb recruits components of the ubiquitination machinery to the Notch receptor thereby facilitating Notch1 ubiquitination at the membrane, which in turn promotes degradation of the intracellular domain circumventing its nuclear translocation and downstream activation of Notch1 target genes.