The 27-base pair (bp) repeats in intron 4 were considered as the source of small RNA that suppressed endothelial nitric oxide synthase (eNOS) expression in endothelial cells. The present study was to investigate the role of polymorphic intronic 27-nucleotide (nt) miRNA and nuclear transcription factors (TFs) in eNOS expression in endothelial cells. The 27-nt or 54-nt miRNA or their mutants (with 3 mutations at 13, 14, and 15th nt for 27-nt mutant and at 13,14,15,40,41,42th nt for 54-nt mutant) were stably transfected and expressed in human aortic endothelial cells (HAECs). Overexpression of 27-nt miRNA and its duplexes and mutants significantly decreased eNOS mRNA level and protein content. Interestingly, more reduction in eNOS protein was observed in the cells overexpressing the mutant miRNAs than the cells overexpressing the wild-type ones for both 27-nt and 54-nt miRNAs. Western blotting showed that transcription factor Ap-1 expression was substantially decreased in the cells that overexpressed either 27-nt or 54-nt miRNAs (both wild-type and mutants). Augmented inhibition of Ap-1 expression was observed in the cells overexpressing the mutant 27-nt miRNA as compared with the wild-type. However, Sp-1 protein expression was significantly suppressed only in the cells overexpressing 54-nt miRNA. Silencing Ap-1 abolished the polymorphic effect of miRNAs both in length and mutation on eNOS expression; while Sp-1 silencing only reversed the polymorphic effect of mutant miRNAs. These results suggested that overexpression of intron 4-based 27-nt miRNAs suppressed eNOS gene expression polymorphically through interactions predominantly with nuclear transcription factors Ap-1 in HAECs.