Background: Restenosis after angioplasty for coronary vascular disease remains a critical problem in cardiovascular medicine. Vascular smooth muscle cell (VSMC) migration and proliferation cause restenosis through neointima formation. Mitochondrial motility is likely necessary for cell proliferation and migration, and is inhibited in microdomains with increased Ca 2+ . The Ca 2+ /calmodulin-dependent kinase II (CaMKII) in mitochondria (mtCaMKII) is proposed to control mitochondrial matrix Ca 2+ uptake through mitochondrial Ca 2+ uniporter (MCU). Thus, we hypothesized that blocking mtCaMKII decreases VSMC migration and neointima formation by decreasing mitochondrial motility. Methods: mtCaMKII was inhibited by expression of the mitochondria-targeted CaMKII inhibitor peptide (CaMKIIN) in a novel transgenic mouse model in smooth muscle only (SM-mtCaMKIIN) or delivered by adenoviral transduction (Ad-mtCaMKIIN). Results: In our models, mtCaMKIIN was detected selectively in mitochondria of VSMC. mtCaMKIIN significantly reduced mitochondrial Ca 2+ current and Ca 2+ content compared to WT in vivo and in vitro. SM-mtCaMKIIN mice showed significantly reduced neointimal area 28 days after endothelial injury (n=8, p<0.05) and fewer proliferating neointimal cells by PCNA staining. In vitro, Ad-mtCaMKIIN mildly reduced VSMC proliferation and mitochondrial ROS production without altering maximal respiration after PDGF treatment. Ad-mtCaMKIIN abolished VSMC migration, as did mitoTEMPO and MCU inhibitor Ru360. Ad-mtCaMKIIN blocked mitochondrial mobility towards the leading edge, while relocation of mitochondria was seen in WT cells 6 h after PDGF treatment. Mitochondrial redistribution was also inhibited by Ru360, but not by mitoTEMPO or cytoplasmic CaMKII inhibition. Mitochondrial fission promotes cell migration. Accordingly, PDGF increased mitochondrial particles in WT VSMC, while mitochondria in Ad-mtCaMKIIN cells were fragmented and unresponsive to PDGF treatment. Conclusions: mtCaMKIIN prevents mitochondrial distribution to the leading edge and reduces VSMC migration and neointima formation. These data suggest mitochondrial Ca 2+ regulation plays an important role in VSMC migration by altering mitochondrial location.