Mitochondria play a central role in cell survival, and apoptotic cell death is associated with morphological changes in mitochondria. Quantification of the morphological and mechanical property changes in brain mitochondria is useful for evaluating the degree of ischemic injury and the neuroprotective effects of various drugs. This study was performed to investigate the changes in brain mitochondria in an 11-vessel occlusion ischemic model treated with magnesium sulfate (MgSO₄), utilizing atomic force microscopy (AFM). Rats were randomly divided into three groups consisting of sham (n=6), global ischemia (GI, n=6), and MgSO₄-treated global ischemia (MgSO₄, n=6). The biophysical properties of brain mitochondria determined from AFM topographic images and adhesion force from force–distance measurements. The mean perimeter of ischemic mitochondria significantly increased to 2,396±541nm (vs. 1,006±318nm in control group, P<0.001). The MgSO₄ treatment during global ischemia reduced the perimeter of ischemic mitochondria (1,127±399nm, P<0.001). The other parameters including length, width and area were significantly different than the GI group. Besides, the adhesion force (23.2±3.9nN) of isolated mitochondria from the MgSO₄ group was close to normal levels (28.5±2.5nN), compared with that of ischemic ones (17.7±3.3nN, P<0.001). To confirm the neuroprotective effects of MgSO₄, we performed Nissl staining. This study suggested that quantitative analysis of mitochondrial changes utilizing AFM could be effective for evaluating neuronal injury and drug effects.