Mice deficient in Notch3 have defects in arterial vascular smooth muscle cell (VSMC) mechanosensitivity, including impaired myogenic responses and autoregulation, and inappropriate VMSC orientation. Experiments were performed to determine if Notch3 is activated by mechanical stimulation and contributes to mechanosensitive responses of VSMCs, including cell realignment. Cyclic, uniaxial stretch (10%, 1 Hz) of human VSMCs caused Notch3 activation, demonstrated by a stretch-induced increase in hairy and enhancer of split 1/hairy-related transcription factor-1 expression, translocation of Notch3 to the nucleus, and a decrease in the Notch3 extracellular domain. These effects were prevented by inhibiting the expression [small interfering (si)RNA] or proteolytic activation of Notch3 {N-(R)-[2-(hydroxyaminocarbonyl)methyl]-4-methylpentanoyl-l-naphthylalanyl-l-alanine-2-aminoethyl amide (TAPI-1; 50 μmol/l) to inhibit TNF-α-converting enzyme (TACE) or N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester (DAPT; 20 μmol/l) to inhibit γ-secretase}. Stretch increased the activity of ROS within VSMCs, determined using dichlorodihydrofluorescein fluorescence. Catalase (1,200 U/ml), which degrades H₂O₂, inhibited the stretch-induced activation of Notch3, whereas in nonstretched cells, increasing H₂O₂ activity [H₂O₂ or manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin] caused activation of Notch3. Stretch increased the activity of TACE, which was prevented by catalase. Stretch-induced activation of p38 MAPK in VSMCs was inhibited either by catalase or by inhibiting Notch3 expression (siRNA). Stretch caused VSMCs to realign perpendicular to the direction of the mechanical stimulus, which was significantly inhibited by catalase or by inhibiting the expression (siRNA) or activation of Notch3 (TAPI-1 or DAPT). Therefore, cyclic uniaxial stretch activates Notch3 signaling through a ROS-mediated mechanism, and the presence of Notch3 is necessary for proper stretch-induced cell alignment in VSMCs. This mechanism may contribute to the physiological role of Notch3 in mediating developmental maturation of VSMCs.