This study aims to investigate the role and mechanism of luteolin in inflammation and phenotypic switch of vascular smooth muscle cells (VSMCs) in an arteriovenous fistula (AVF) model, for providing a potential agent for the prevention and therapy of AVF neointimal hyperplasia. In vivo, an AVF model is created in Sprague Dawley rats. In vitro, rat VSMCs are treated with platelet-derived growth factor-BB (PDGF-BB) to induce the phenotypic switch of VSMCs. Histological AVF changes are analyzed using hematoxylin-eosin. Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) are utilized to detect prostaglandin-endoperoxide synthase 2 (PTGS2) expression. In vivo, luteolin inhibits neointima formation and reduces vimentin, α-SMA, MCP-1, MMP-9, TNF-α, and IL-6 levels. In vitro, under PDGF-BB treatment, luteolin inhibits proliferation and migration and reduces TNF-α, vimentin, α-SMA, MCP-1, MMP-9, and IL-6 levels in VSMCs. In rat AVF tissues, PTGS2 expression is increased. Luteolin inhibits PTGS2 expression in vivo and in vitro. PTGS2 overexpression reverses the role of luteolin in extracellular matrix protein expression, proliferation, inflammation, and migration in VSMCs treated with PDGF-BB. Altogether, in the AVF, luteolin inhibits proliferation, migration, the phenotypic switch of VSMCs, neointima formation, and the inflammatory response through inhibiting PTGS2 expression.