Hyperhomocysteinemia (Hhcy) is associated with atherosclerosis, stroke and dementia. Hcy causes extracellular matrix (ECM) remodeling by the activation of matrix metalloproteinase-9 (MMP-9) in part by inducing redox signaling and modulating the intracellular calcium dynamics. Calpains are the calcium-dependent cysteine proteases which are implicated in mitochondrial damage via oxidative burst. Mitochondrial abnormalities have been identified in Hhcy. The mechanism of Hcy-induced ECM remodeling by MMP-9 activation via mitochondrial pathway is largely unknown. We report a novel role of calpains in mitochondrial-mediated MMP-9 activation by Hcy in cultured rat heart microvascular endothelial cells (MVEC). Our observations suggested that calpain regulates Hcy-induced MMP-9 expression and activity. We showed that Hcy activates calpain-1 but not the calpain-2 in calcium-dependent manner. Interestingly, the enhanced calpain activity was not mirrored by the decreased levels of its endogenous inhibitor calpastatin. We presented the evidence that Hcy induces the translocation of active calpain from cytosol to mitochondria leading to MMP-9 activation, in part, by causing intramitochondrial oxidative burst. Furthermore, studies with pharmacological inhibitors of calpain (calpeptin, calpain-1 inhibitor) and ERK (PD98059) and mitochondrial uncoupler, FCCP suggested that calpain and ERK-1/2 are the major events within Hcy/MMP-9 signal axis and intramitochondrial oxidative stress regulates MMP-9 via ERK-1/2 signal cascade. Taken together these findings determine the novel role of mitochondrial translocation of calpain-1 in MMP-9 activation during Hhcy, in part, by increasing mitochondrial oxidative tress.