BACKGROUND Previously, we showed that the 27nt repeat polymorphism in endothelial nitric oxide synthase (eNOS) intron 4 was associated with altered eNOS mRNA and protein levels, nitric oxide (NO) production and vascular disease risk; the 27-nt repeats had a cis-acting role in eNOS promoter function. In the present study, we investigated nuclear protein that binds the 27nt repeat and mediates eNOS expression. RESULTS Using 5'-biotin-labeled 27nt DNA duplex and streptavidin-agarose beads pull-down assay and mass spectrometry, we identified that nuclear beta-actin was one of the major 27nt binding proteins. Using the pGL3 reporter vectors containing the 5x27nt repeats as an enhancer in an in vitro transcription assay, we found that exogenous beta-actin significantly increased reporter gene transcription efficiency. The beta-actin's upregulating effect was compromised when exogenous 27nt RNA duplex was added. Furthermore, the eNOS expression was reduced when beta-actin gene was silenced by specific siRNA, and actin overexpression upregulated eNOS expression >3-fold. CONCLUSIONS Our data demonstrate that beta-actin as a transcription factor stimulates eNOS expression; and the transcriptional effect appears to be 27nt-dependent. Our findings represent a novel molecular mechanism regulating eNOS expression, which could potentially lead to discoveries of eNOS specific pharmaceutical agents, eg, active peptides, with clinical applications.