Both mitochondrial aspartate aminotransferase (mAST) and cytosolic aspartate aminotransferase (cAST) are important components in the malate–aspartate shuttle – one of the two types of NADH shuttles in cells. A major goal of our current study was to determine specifically the roles of cAST in maintaining the [Ca2+]i, mitochondrial membrane potential and the survival of BV2 microglia by applying molecular approach to modulate the cAST levels. Our study found that decreased cAST by cAST siRNA can lead to significant increases in the [Ca2+]i, mitochondrial depolarization and apoptosis of BV2 microglia. The cAST siRNA-induced mitochondrial depolarization can be significantly attenuated by an inhibitor of calpain. We further found that the cAST siRNA-induced apoptosis can be prevented by the calpain inhibitor. Collectively, our study suggests that decreased cAST induces calpain activation by increasing the [Ca2+]i of BV2 microglia, resulting in mitochondrial depolarization and cell death. Moreover, our data suggest that decreased cAST may produce these pathological effects by malate–aspartate shuttle-independent pathways.