Osteopontin (OPN) is involved in the regulation of calcium precipitation in the brain pathology including ischemia. A 3-Nitropropionic acid (3NP) irreversibly inhibits mitochondrial complex II in the electron transport chain, with subsequent loss of transmembrane potential and calcium entry into the mitochondria. The present study examined the 3NP-induced calcium elevation in mitochondria and OPN expression in the 3NP-lesioned striatum. Rats were subcutaneously injected 3NP (15 mg÷kg) every other day for six weeks. Histological analysis, including the Hematoxylin-Eosin, Nissl, and Alizarin Red S stainings, was performed to examine the neurotoxic effects of 3NP. The expression of OPN in the striatum of 3NP-treated rats was investigated with immunohistochemistry and immunoelectron microscopy. In the striatal lesions, extensive loss of neurons and white matter bundles was detected. OPN was mainly detected in the penumbra region of the 3NP lesion. Scattered OPN expression was colocalized in the striatal neurons. After Alizarin Red S staining, the increase of calcium deposition was detected in the striatal lesions. In the electron microscopic analysis, the localization of OPN was clearly observed in the ultrastructure of mitochondria by immunoperoxidase and immunogold-silver staining techniques. Taken together, present findings suggest that calcium-induced mitochondrial swelling is highly associated with OPN expression. Thus, striatal calcium accumulation may be derived from 3NP-induced alteration in mitochondrial calcium homeostasis and pathologically associated with the induction of OPN protein.