Vascular smooth muscle cell (VSMC) phenotypic modulation is a key factor in vascular pathology. We have investigated the role of Notch receptor signaling in controlling human vascular smooth muscle cell (hVSMC) differentiation in vitro and established a role for cyclic strain-induced changes in Notch signaling in promoting this phenotypic response. The expression of alpha-actin, calponin, myosin, and smoothelin was examined by performing immunocytochemistry, Western blot analysis, and quantitative real-time PCR in hVSMCs cultured under static conditions after forced overexpression of constitutively active Notch 1 and 3 receptors, inhibition of endogenous Cp-binding factor 1 (CBF-1)/recombination signal sequence-binding protein-Jkappa (RBP-Jkappa) signaling, and exposure to cyclic strain using a Flexercell Tension Plus unit. Overexpression of constitutively active Notch intracellular (IC) receptors (Notch 1 IC and Notch 3 IC) resulted in a significant downregulation of alpha-actin, calponin, myosin, and smoothelin expression, an effect that was significantly attenuated after inhibition of Notch-mediated, CBF-1/RBP-Jkappa-dependent signaling by coexpression of RPMS-1 (Epstein-Barr virus-encoded gene product) and selective knockdown of basic helix-loop-helix factors [hairy enhancer of split (HES) gene and Hes-related transcription (Hrt) factors Hrt-1, Hrt-2, and Hrt-3] using targeted small interfering RNA. Cells cultured under conditions of defined equibiaxial cyclic strain (10% strain, 60 cycles/min, 24 h) exhibited a significant reduction in Notch 1 IC and Notch 3 IC expression concomitant with a significant increase in VSMC differentiation marker expression. Moreover, this cyclic strain-induced increase was further enhanced after inhibition of CBF-1/RBP-Jkappa-dependent signaling with RPMS-1. These findings suggest that Notch promotes changes in hVSMC phenotype via activation of CBF-1/RBP-Jkappa-dependent pathways in vitro and contributes to the phenotypic response of VSMCs to cyclic strain-induced changes in VSMC differentiation.