Background/Aims: Estrogen has been reported to have beneficial effects on vascular biology through direct actions on endothelium. Together with transcription factors, miRNAs are the major drivers of gene expression and signaling networks. The objective of this study was to identify a com-prehensive regulatory network (miRNA-transcription factor-downstream genes) that controls the transcriptomic changes observed in endothelial cells exposed to estradiol. Methods: miR-NA/mRNA interactions were assembled using our previous microarray data of human umbilical vein endothelial cells (HUVEC) treated with 17ß- Estradiol (E2) (1 nmol/lL, 24 h). miRNA--mRNA pairings and their associated canonical pathways were determined using Ingenuity Pathway Analysis software. Transcription factors were identified among the miR-NA-regulated genes. Transcription factor downstream target genes were predicted by consensus transcription factor binding sites in the promoter region of E2-regulated genes by using JASPAR and TRANSFAC tools in Enrichr software. Results: miRNA--target pairings were filtered by using differentially expressed miRNAs and mRNAs characterized by a regulatory relationship accord-ing to miRNA target prediction databases. The analysis identified 588 miRNA--target interactions between 102 miRNAs and 588 targets. Specifically, 63 up-regregulated miRNAs interacted with 295 down-regregulated targets, while 39 down-regregulated miRNAs were paired with 293 up-regregulated mRNA targets. Functional characterization of miRNA/mRNA association analy-sis highlighted hypoxia signallignaling, integrin, ephrin receptor signaling, and regulation of actin-based motility by Rho among the canonical pathways regulated by E2 in HUVEC. Tran-scription factors and downstream genes analysis revealed a total of eight networks, including those mediated by JUN and REPIN1, which are associated with cadherin binding and cell adhe-sion molecule binding pathways. Conclusion: This study identifies regulatory networks obtained by integrative microarray analysis and provides additional insights into the way estradiol could regulate endothelial function in human endothelial cells.