Sirolimus is a potent immunosuppressive agent and has an anti-atherosclerotic effect through its anti-proliferative property. The present study was undertaken to investigate the effect of Sirolimus on intracellular cholesterol homeostasis in human vascular smooth muscle cells (VSMCs) in the presence of inflammatory cytokine, IL-1. We explored the effect of Sirolimus on the lipid accumulation of VSMCs in the presence of IL-1, using Oil Red O staining and quantitative measurement of intracellular cholesterol. The effect of Sirolimus on the gene and protein expression of lipoprotein receptors and adenosine triphosphate (ATP) binding cassettes (ABCA1 and ABCG1) was examined by real-time PCR and western blotting, respectively. Furthermore, the effect of Sirolimus on cholesterol efflux from VSMCs in the presence or absence of IL-1 was also investigated using [3H]-labelled cholesterol efflux. Finally, we examined the effect of Sirolimus on the production of inflammatory cytokines in VSMCs using ELISA. Sirolimus reduced intracellular lipid accumulation in VSMCs mediated by IL-1 possibly due to the reduction of expression of LDL and VLDL receptors. Sirolimus increased cholesterol efflux from VSMCs and overrode the suppression of cholesterol efflux induced by IL-1. Sirolimus also increased ABCA1 and ABCG1 genes expression, even in the presence of IL-1. We further confirmed that Sirolimus inhibited mRNA and protein expression of inflammatory cytokines IL-6, TNF, IL-8, and MCP-1. Inhibition of lipid uptake together with increasing cholesterol efflux and the inhibition of inflammatory cytokines are all important aspects of the anti-atherosclerotic effects of Sirolimus on VSMCs.