Recent studies have suggested a protective role of hsp27 against atherosclerosis and transplant graft vasculopathy. Here we have investigated the effects of over-expression of wild-type hsp27 and its phosphorylation mimics on proliferation of human endothelial cells (ECs) and smooth muscle cells (SMCs). ECs and SMCs cultured from human blood vessels or cells lines (human microvascular endothelial cell line and human telomerase reverse transcriptase subunit SMC) were infected with adenovirus containing DNA from wild-type hsp27, hyper-phosphorylated hsp27 mimic (3D hsp27), hypo-phosphorylated hsp27 mimic (3A hsp27) or anti-sense hsp27, and proliferation measured over the next 5 days. Protein extracts from infected cells were subjected to proteomic analysis using 2-D DIGE. Over-expression of 3D hsp27 and anti-sense hsp27 but not 3A hsp27 mimic caused significant inhibition of proliferation of ECs and SMCs. Proteomic analysis focussed on proteins that were significantly down-regulated by the 3D hsp27 mutant. The cell cycling proteins stathmin, cofilin and ubiquitination enzymes fullfilled these criteria. 1-D Western blots of infected human microvascular endothelial cell line and human telomerase reverse transcriptase subunit SMC confirmed down-regulation of stathmin, cofilin and ubiquitination enzymes by 3D hsp27. The phosphorylation status of hsp27 is an important regulator of proliferation of human vascular ECs and SMCs; possibly contributing to cardiovascular protection.