Tumor necrosis factor-α (TNFα) is a proinflammatory cytokine that is closely linked to the development of cardiovascular disease. TNFα activates NADPH oxidase 1 (Nox1) and reactive oxygen species (ROS), including superoxide (O), production extracellularly is required for subsequent signaling in vascular smooth muscle cells (VSMCs). Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase that is activated by oxidation of associated thioredoxin. The role of ASK1 in Nox1-mediated signaling by TNFα is poorly defined. We hypothesized that ASK1 is required for TNFα receptor endocytosis and subsequent inflammatory TNFα signaling. We employed a knockdown strategy to explore the role of ASK1 in TNFα signaling in VSMCs. siRNA targeting ASK1 had no effect on TNFα-induced extracellular O production. However, siASK1 inhibited receptor endocytosis as well as phosphorylation of two endocytosis-related proteins, dynamin1 and caveolin1. Intracellular O production was subsequently reduced, as were other inflammatory signaling steps including NF-κB activation, IL-6 production, inducible nitric oxide synthase and VCAM expression, and VSMC proliferation. Prolonged exposure to TNFα (24 h) increased tumor necrosis factor receptor (TNFR) subtype 1 and 2 expression, and these effects were also attenuated by siASK1. ASK1 coimmunoprecipitated with both Nox1 and the leucine rich repeat containing 8A anion channel, two essential components of the TNFR1 signaling complex. Activation of ASK1 by autophosphorylation at Thr845 occurs following thioredoxin dissociation, and this requires the presence of Nox1. Thus, Nox1 is part of the multiprotein ASK1 signaling complex. In response to TNFα, ASK1 is activated by Nox1-derived oxidants, and this plays a critical role in translating these ROS into a physiologic response in VSMCs. Apoptosis signal-regulating kinase 1 (ASK1) drives dynamin1 and caveolin1 phosphorylation and TNFα receptor endocytosis. ASK1 modulates TNFα-induced NF-κB activation, survival, and proliferation. ASK1 and NADPH oxidase 1 (Nox1) physically associate in a multiprotein signaling complex. Nox1 is required for TNFα-induced ASK1 activation.