Cardiac fibrosis is characterized by the excessive deposition of extracellular matrix, including fibrillar collagens, proteoglycans, and fibronectin. This process is energy‐intensive for cardiac fibroblasts, however it remains unclear how these energy metabolic requirements are met. Liver fibrosis, and in particular collagen synthesis, has been shown to be dependent on glutaminolysis, in which glutamine is converted to glutamate by the enzyme glutaminase (GLS) with further conversion to a‐ketoglutarate to fuel oxidative metabolism via the tricarboxylic acid cycle, but a role for glutaminolysis in cardiac fibrosis remains to be demonstrated. We previously showed that scleraxis (Scx) is a transcription factor that is critically required for the conversion of cardiac fibroblasts to myofibroblasts, but its potential role in glutaminolysis in these cells is unknown. We report here that freshly‐isolated rat cardiac fibroblasts (P0 – unpassaged) passaged twice to become myofibroblasts (P2) show concomitant up‐regulation of Scx by 5‐fold and GLS1 by 4‐fold in P2 versus P0. Scx over‐expression induced a 20‐fold increase in GLS1 expression. To determine whether GLS1 expression is dependent on Scx, we examined GLS1 expression in activated mouse cardiac fibroblasts (P1) isolated from wild type or Scx knockout mice. Scx knockout attenuated GLS1 expression by almost 90%. TGFb is a potent inducer of cardiac fibrosis that both dramatically up‐regulates Scx expression, and requires Scx for many of its downstream effects. TGFb treatment of wild type mouse P1 activated fibroblasts doubled the expression of GLS1, but had no effect in Scx KO cells. In silico analysis revealed four putative Scx binding sites in the GLS1 promoter. While TGFb significantly up‐regulated expression of the myofibroblast marker periostin in cardiac fibroblasts, both scleraxis knockout and the glutaminase inhibitor CB‐839 attenuated this induction, indicating a failure to convert to myofibroblasts. These findings suggest that Scx regulates GLS1 expression to facilitate increased energy metabolism during cardiac fibroblast to myofibroblast conversion.Support or Funding InformationCanadian Institutes of Health Research (PJT‐162422).