Aim Based on the signaling pathway of autophagosome formation to discuss the protective effect and mechanism of Tanshinone ⅡA on endothelial cell oxidative stress injury. Methods Culturing EA.hy926 cells in vitro, then randomly dividing these cells into normal group, model group, tanshinone ⅡA group, tanshinone ⅡA and model group, 3-MA group, model and 3-MA group, tanshinone ⅡA, model and 3-MA group. Using colorimetric method to test the cells’ MDA content and SOD activity of oxidative stress injury. Western blot detecting the expressions of cells’ autophagosome formation signaling pathway related protein. Results Compared with normal group, the MDA content is increased (P<0.01), the SOD activity is decreased (P<0.01), and the content of LC3-Ⅰ/LC3-Ⅱ protein is increased (P<0.01) in model group. There is no obvious change of LC3-Ⅰ/LC3-Ⅱ protein content (P＞0.05) in 3-MA group compared with normal group. Compared with model group, the MDA content is decreased (P<0.01), the SOD activity is increased (P<0.01) and the content of LC3-Ⅰ/LC3-Ⅱ protein is increased (P<0.01) in tanshinone ⅡA and model group. The MDA content is increased (P<0.01), the SOD activity is decreased (P<0.01), and the content of LC3-Ⅰ/LC3-Ⅱ protein is decreased (P<0.01) in model and 3-MA group compared with model group. Compared with tanshinone ⅡA and model group, the MDA content is increased (P<0.01), the SOD activity is decreased (P<0.01), and the content of LC3-Ⅰ/LC3-Ⅱ protein is decreased (P<0.01) in tanshinone ⅡA, model and 3-MA group. Compared with normal group, the expression of Atg3, Atg4b, Atg7 are increased significantly in model group (P<0.05 or P<0.01). Compared with model group, the expression of Atg3, Atg7 , Atg5-Atg12 are increased significantly in tanshinone ⅡA and model group (P<0.05 or P<0.01). Compared with tanshinone ⅡA and model group, the expression of Atg3, Atg7 , Atg5-Atg12 are decreased significantly in tanshinone ⅡA, model and 3-MA group (P<0.05 or P<0.01). Conclusion Tanshinone ⅡA may regulate the proteins of signaling pathway of autophagosome formation as well as the Atg12-Atg5 pathway and LC3-PE pathway of EA.hy926 cell to play its protective biological activities for EA.hy926 cells against oxidative stress damage, then prevent and treat the development of As.