Previously it was reported that the 27-nucleotide(nt) repeats in intron 4 of endothelial nitric syanthase (eNOS) were the source of 27-nt microRNA, which play an important role in regulation of eNOS expression. In order to further study the molecular mechanisms in regulation of eNOS gene expression and endothelial cell proliferation by the 27-nt microRNA, it was constructed the 27-nt microRNA highly expression plasmid, which was transferred into HUVEC with lipofectamine. The level of eNOS protein and mRNA, as well as Sp1 and Ap-1,were measured by Western blot and RT-PCR, respectively. The proliferation of the HUVEC was analyzed by MTT. The results showed that the 27-nt microRNA can significantly decrease eNOS mRNA level by 94.6% (0.015 ± 0.006 vs 0.277 ± 0.012, P < 0.01) and inhibit its protein expression by 94.9%(0.012 ± 0.005 vs 0.237 ± 0.010, P < 0.01); Sp1 and Ap1 protein were significantly decreased by 14.0% (0.860 ± 0.013 vs 1.000 ±0.015, P < 0.05) and by 22.0% (0.780 ± 0.033 vs 1.000 ± 0.052, P < 0.05), compared with control, respectively. The growth rate of HUVEC treated with the 27-nt microRNA high expression was significantly decreased, particularly the inhibition in the cell lines transfected with double-length and mutant of the 27-nt microRNA plasmid, by which the doubling-time of growth was increased by 49.4% (29.22 ± 0.25 vs 19.55 ± 0.19, P < 0.05), compared with control. The data suggested that intronic 27-nt microRNA significantly inhibit the eNOS expression and the proliferation of HUVEC, by the time the expression of transcription factors Sp-1 and Ap-1 were altered, strongly suggesting that 27-nt microRNA and transcription factors cooperatively regulate the expression of related genes, consequently the proliferation of HUVEC. Data from the present study may serve as one model of the critical mechanisms through which the intronic microRNA and transcription factor synergisticly were involved in the auto-regulation of disease-related gene expression.