Adventitial fibroblasts (AFs) are the main cell type in the adventitia, however, their role in atherosclerosis remains unclear. We have investigate the role of AFs in atherosclerotic lesion formation by comparing the characteristics of AFs from apoE(−/−) to C57BL/6 mice. A minority of AFs from apoE(−/−) mice expressed α-SM-actin, but no α-SM-actin-positive cells were found in AFs from C57BL/6 mice. The content of total collagens, and the mRNA levels of collagen I and collagen III in AFs of apoE(−/−) mice, were higher than in C57BL/6 mice. AFs from apoE(−/−) mice proliferate and migrate faster, and synthesized more TGF-β1, MCP-1, and PDGF-b AFs from apoE(−/−) mice have the characteristics of transdifferentiation into myofibroblasts, including enhanced proliferation and migration, along with synthesis of collagens and cytokines compared to AFs from C57BL/6 mice. The histological and functional characteristics of AFs may contribute to early atherosclerotic lesion formation.