Aim We previously reported that endothelial nitric oxide synthase （eNOS） intron 4 of 27 bp repeat promoter to produce the corresponding 27-nt miRNA, which regulated expression of eNOS. This study was to investigate an intronic miRNA regulated expression of the human eNOS gene and proliferation of endothelial cells by mechanism related to the transcription factor AP1. Methods The endothelial cells proliferation inhibition rate was detected by MTT assay. Migration ability of endothelial cells was measured by scratch wound model in vitro. The protein expression level of AP1 and eNOS were detected by Western Blot. Results Overexpression of 27-nt miRNA significantly inhibited the endothelial cells proliferation （P<0.05）. Overexpression of 27-nt miRNA significantly inhibited HUVEC migration （P<0.05）. Overexpression of 27-nt miRNA significantly decreased AP1 and eNOS protein expression （P<0.05）. Overexpression of 27-nt miRNA significantly decreased eNOS protein expression by the transcription factor AP1. Conclusions The 27nt-miRNA suppresses eNOS gene expression and AP1 expression in endothelial cells. 27-nt miRNA regulated expression of the eNOS by the transcription factor AP1.