The nucleus of the endothelial cell contains large amounts of high-mobility group box protein 1 (HMGB1), a cytokine mediator of inflammation, and the endothelium may be a crucial source of HMGB1 during the inflammatory response. Therefore, the downregulation of HMGB1 expression by RNA interference (RNAi) may decrease inflammatory activity. The aim of this study was to investigate the possible mechanism of action and the effect of HMGB1 on homeobox A9 (HOXA9) and E-selectin expression. Recombinant human full-length HMGB1 was cloned by PCR amplification from human umbilical vein endothelial cells (HUVECs) and then subcloned into a pcDNA‑3.1-myc-his-B vector. Specific short hairpin RNAs (shRNAs) for the HMGB1 target sequence and a scrambled sequence were designed, synthesized and cloned into a pRNA‑U6.1/Neo vector. Specific small interfering RNAs (siRNAs) for the HOXA9 target sequence were commercially prepared and the expression levels of HMGB1, HOXA9, intracellular adhesion molecule 1 (ICAM1), vascular cell adhesion molecule 1 (VCAM1) and E-selectin were detected using real-time PCR and western blot analysis. The expression of the full-length HMGB1 gene and protein was verified in HUVECs. The shRNA for HMGB1 and siRNA for HOXA9 successfully decreased the expression levels of HMGB1 and HOXA9, respectively. ICAM1, VCAM1 and E-selectin were downregulated through HMGB1 interference in HUVECs, and HMGB1 shRNA decreased E-selectin expression by HOXA9. These results demonstrated the potential use of specific siRNA targeting HMGB1 expression for the development of novel therapeutic agents for inflammatory disorders.