Objective— This study was designed to carry out the characterization of stem cells within the adventitia and to elucidate their functional role in the pathogenesis of vein graft atherosclerosis. Approach and Results— A mouse vein graft model was used to investigate the functional role of adventitial stem/progenitor cells on atherosclerosis. The adventitia of vein grafts underwent significant remodeling during early stages of vessel grafting and displayed markedly heterogeneous cell compositions. Immunofluorescence staining indicated a significant number of stem cell antigen-1–positive cells that were closely located to vasa vasorum. In vitro clonogenic assays demonstrated 1% to 11% of growing rates from adventitial cell cultures, most of which could be differentiated into smooth muscle cells (SMCs). These stem cell antigen-1–positive cells also displayed a potential to differentiate into adipogenic, osteogenic, or chondrogenic lineages in vitro. In light of the proatherogenic roles of SMCs in atherosclerosis, we focused on the functional roles of progenitor-SMC differentiation, in which we subsequently demonstrated that it was driven by direct interaction of the integrin/collagen IV axis. The ex vivo bioreactor system revealed the migratory capacity of stem cell antigen-1–positive progenitor cells into the vessel wall in response to stromal cell-derived factor-1. Stem cell antigen-1–positive cells that were applied to the outer layer of vein grafts showed enhanced atherosclerosis in apolipoprotein E–deficient mice, which contributed to ≈30% of neointimal SMCs. Conclusions— We demonstrate that during pathological conditions in vein grafting, the adventitia harbors stem/progenitor cells that can actively participate in the pathogenesis of vascular disease via differentiation into SMCs.