Transforming growth factor-β (TGF-β) plays an important role in vascular homeostasis through effects on vascular smooth muscle cells (SMC). Fine-tuning of TGF-β signaling occurs at the level of ALK receptors or Smads, and is regulated with cell type specificity. Our goal was to understand TGF-β signaling in regulating SMC differentiation marker expression in human SMC. Activation of Smads was characterized, and loss-and gain-of-function reagents used to define ALK pathways. In addition, Smad-independent mechanisms were determined. TGF-β type I receptors, ALK1 and ALK5, are expressed in human SMC, and TGF-β1 phosphorylates Smad1/5/8 and Smad2/3 in a time-and dosage-dependent pattern. ALK5 activity, not bone morphogenetic protein type I receptors, is required for Smad phosphorylation. Endoglin, a TGF-β type III receptor, is a TGF-β1 target in SMC, yet endoglin does not modify TGF-β1 responsiveness. ALK5, not ALK1, is required for TGF-β1-induction of SMC differentiation markers, and ALK5 signals through an ALK5/Smad3-and MAP kinase-dependent pathway. The definition of the specific signaling downstream of TGF-β regulating SMC differentiation markers will contribute to a better understanding of vascular disorders involving changes in SMC phenotype.