Background and Objectives: Previously, we have demonstrated that heat shock protein 27 (Hsp27) is significantly less expressed in human carotid atherosclerotic lesions. We tried to investigate the role of HSP27 in oxidative stress, especially in the maintenance of extracellar matrix in atherosclerotic lesions. Methods and Results: HSP27 is abundantly expressed in the cytoplasm of smooth muscle cells(SMCs) from human internal mammary artery (IMA) and early atherosclerotic lesions from ApoE-/- mice. In advanced atherosclerotic lesions in human carotid arteries and high fat high cholesterol-fed ApoE-/- mice, the intracellular level of HSP27 expression in SMCs is decreased and the protein appeared extracellularly surrounding SMCs and macrophages, especially in deep intima. When SMCs isolated from human aortic SMCs or human umbilical cord were transduced with adenoviral vector containing wild type HSP27, cell death caused by oxidized LDL was significantly reduced and collagens - type I, III and IV- expression was significantly enhanced compared to those from cells transduced with control virus or virus containing mutant HSP27 in hypoxic condition. Production of tissue plasminogen activator and p-selectin also was reduced by overexpression of wtHSP27. Interestingly, HSP27 from IMAs appeared as monomer whereas that from carotid artery enarterectomy specimen(CAES) showed both monomer and oligomer. HSP27 in core regions from CAES forms exclusively oligomer. More interestingly, HSP27 at Ser78 and Ser82 was heavily phosphorylated in momomer from both IMA and normal looking area of CAES but was not or mildly phosphorylated in oligomer from CAES. On the other hand, HSP27 at Ser15 was not phosphorylated in monomer from IMA but was pjosphorylated manily in oligomer from CAES. Conclusions: HSP27 prevents smooth muscle cell death by modulating in part the differential expression of collagens, Bcl2 family protein, and other ECM related proteins. HSP27 may function differently based on its phosphorylation at different sites of the protein. In conclusion, HSP27 may play a major role in homeostasis of arterial wall by balancing ECM and constituent cells and may be used to predict the degree of progression of atherosclerosis by analyzing its phosphorylation pattern.