Intercellular signaling mediated by Notch receptors is essential for proper cardiovascular development and homeostasis. Notch regulates cell fate decisions that affect proliferation, survival, and differentiation of endothelial and smooth muscle cells. It has been reported that Jagged1–Notch interactions may participate in endocardial cushion formation by inducing endothelial-to-mesenchymal transformation. Here, we show that Notch directly regulates expression of the mesenchymal and smooth muscle cell marker smooth muscle α-actin (SMA) in endothelial and vascular smooth muscle cells via activation of its major effector, CSL. Notch/CSL activation induces SMA expression during endothelial-to-mesenchymal transformation, and Notch activation is required for expression of SMA in vascular smooth muscle cells. CSL directly binds a conserved cis element in the SMA promoter, and this consensus sequence is required for Notch-mediated SMA induction. This is the first evidence of the requirement for Notch activation in the regulation of SMA expression.