The aim of the study was to determine the importance and possible mechanism of NAD (P)H oxidase subunits P (superscript 22phox) involved in human umbilical endothelial cell lines ECV-304 aging by special short interference RNA (siRNA). Three siRNAs targeting p22phox were designed and synthesized in vitro, which were used to transfect ECV-304 cultured in vitro for selecting the most powerful and most suitable transfection concentration and time. The cell line ECV-304 was divided into three groups: control group, angiotensinⅡ(AngⅡ) group, siRNA group, and AngⅡ+siRNA group. Cell aging was identified by b-gal stain. Reactive oxygen species (ROS) and NO level in cells and medium were measured. RT-PCR and Western blot were used to analyze mRNA and protein expression of NAD(P)H oxidase subunit p22phox. Among the 3 siRNAs, siRNA-1 was the most powerful on gene silence with 50 nmol/L transfection concentration at 24 h and 36 h. The number of positive cells stained by b-gal were increased in ECV-304 stimulated with AngⅡ, and p22phox mRNA and protein expression were increased in aging ECV-304 stimulated with AngⅡ, which had lower NO and higher ROS. Compared with AngⅡgroup, ROS level was decreased and NO level was increased in AngⅡ+siRNA group with decreased aging level. The result of the present study suggested that siRNA could induce NAD(P)H oxidase subunit p22phox gene silence, AngⅡcould induce ECV-304 aging cultured in vitro, and the possible pathway of endothelial cell aging is that AngⅡupregulates p22phox expression, and then enhances the cell ROS level.