Objective:To investigate the reverse effect of astragaloside Ⅳ (As-Ⅳ) on angiotensin Ⅱ (Ang Ⅱ)-induced mitochondrial dysfunction of vascular smooth muscle cells (VSMCs) in rats. Methods: Cultured VSMCs were divided into the 24 h control group, the Ang Ⅱ treated for 24 h group, the 48 h control group, the Ang Ⅱ treated for 48 h group,and the As-Ⅳ treated group. After treatment, the 24 h control group and the Ang Ⅱ treated for 24 h group were tested for mitochondrial function by Extracellular Flux Analyzer and mitochondrial ATP production. The 48 h control group, the Ang Ⅱ treated for 48 h group,and the As-Ⅳ treated group were tested for mitochondrial function, mitochondrial ATP production, mitochondrial morphology by transmission electron microscope, and reactive oxygen species (ROS) production by confocal microscopy and Mn-SOD activity. Results: Compared with the 24 h controls group, oxygen consumption rates (OCRs) and mitochondrial ATP production decreased in the Ang Ⅱ treated for 24 h group (P ≤ 0.05); Compared with the 48 h controls group, the Ang Ⅱ treated for 48 h group showed a decrease of OCRs and mitochondrial ATP production (P ≤ 0.05), swollen and vacuolization of mitochondrial morphology with almost wash-out cristae, a decrease of Mn-SOD activity (P ≤ 0.05) and a increase of mtROS level (P ≤ 0.05). The As-Ⅳ treatment group showed significantly increased OCRs, mitochondrial ATP production (P ≤ 0.05) and Mn-SOD activity (P ≤ 0.05), decreased mtROS level (P ≤ 0.05), and reduced damage of mitochondrial morphology compared with the Ang Ⅱ treated for 24 h group. Conclusion: This study demonstrates that As-Ⅳ could reverse Ang Ⅱ induced mitochondrial dysfunction of VSMCs by enhancing Mn-SOD activity to decrease mtROS production, further alleviating the damage of mitochondrial morphology and increasing OCRs and mitochondrial ATP production.