When the heart is injured quiescent fibroblasts differentiate into contractile, synthetic myofibroblasts. Initially fibrosis is reparative, but when chronic it becomes maladaptive and contributes to heart failure progression. Cytosolic Ca2+ (cCa2+) signaling is reported to be necessary for myofibroblast transdifferentiation, yet the role of mitochondrial Ca2+ (mCa2+) exchange has not been explored. cCa2+ signaling is rapidly integrated into the mitochondrial matrix via the mCa2+ uniporter channel (MCUc). To examine the contribution of mCa2+ in cardiac fibrosis, we generated conditional, fibroblast-specific Mcu knockout mice (KO) to ablate mCa2+ uptake. KO and control mice were subjected to myocardial infarction and cardiac function was examined by echocardiography. Loss of mCa2+ uptake worsened left ventricular function and increased fibrosis. To examine the cellular mechanisms responsible for the increased fibrosis we isolated mouse embryonic fibroblasts (MEFs) from Mcufl/fl mice and deleted Mcu with Cre-adenovirus. When challenged with pro-fibrotic ligands (TGF-β and AngII), Mcu-/- MEFs exhibited decreased mCa2+ uptake and enhanced cCa2+ transient amplitude. Loss of Mcu promoted myofibroblast differentiation, including increased α-SMA expression and enhanced contractile function. Mcu-/- MEFs were more glycolytic with increased phosphorylation (inactivation) of pyruvate dehydrogenase. Genetic activation of glycolysis, with a ‘glyco-high’ mutant construct, was sufficient to promote myofibroblast differentiation in WT fibroblasts. Conversely, genetic inhibition of glycolytic flux ablated the enhanced differentiation observed in Mcu-/- fibroblasts. Our results suggest that alterations in mCa2+ uptake and bioenergetic pathways are necessary for myofibroblast differentiation. Thus, energetic signaling represents a novel therapeutic target to impede HF progression and other progressive fibrotic diseases.