Abstract Objective Smooth muscle cells (SMCs) death promotes atherosclerotic lesion necrosis and plaque destabilization. We investigated the potential mechanisms of rat SMCs death in response to excess free cholesterol (FC). Methods and results Rat aortic SMCs were incubated with “water soluble cholesterol” and acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor Sandoz58035 to establish FC-overloading cell model. Disruption of mitochondrial network and endoplasmic reticulum (ER) was observed after 12 h incubation by transient transfection. After treated for 24 h, enhanced cell death was noted as detected by propidium iodide (PI) staining/flow cytometry ( P < 0.001 vs. control). SMCs death was associated with markedly decreased mitochondrial transmembrane potential (Δ φ m), as well as upregulation of cellular reactive oxygen species (ROS) and ER stress. We also investigated possible signaling pathways involved in excess FC-initiated cell death and found that unfolded protein response (UPR) was activated, with increased cellular Bax expression and release of mitochondrial cytochrome c. Conclusion Our findings suggested that FC-overloading might trigger SMCs death. Both ER- and mitochondria-based signals might be implicated in these lethal events.