AbstractMetabolic dysregulations have emerged as a major mediator of cardiovascular disorders and fibrotic diseases. Metabolic reprogramming contributes a lot to cardiac fibroblast activation and cardiac fibrosis post-myocardial infarction (MI), yet the mechanism remains incompletely understood. Our work aimed to determine whether or not glycolytic reprogramming, regulated by phosphofructokinase-2/fructose-2,6-bisphosphatase 3 (PFKFB3), is a therapeutic target for alleviating post-MI cardiac fibrosis. Here, we showed that cardiac fibroblasts displayed cell energy phenotype toward augmented glycolysis in response to transforming growth factor-beta 1 (TGF-β1), evidenced by significant extracellular acidification rate (ECAR) increase and lactate accumulation. The expression of glycolytic enzyme PFKFB3, a master activator of glycolysis, was up-regulated in TGF-β1-treated cardiac fibroblasts and in cardiac fibroblasts of post-MI mice. Pharmacological inhibition of PFKFB3 by 3PO diminished TGF-β1-mediated profibrotic phenotypes, attenuated cardiac fibrosis, and preserved cardiac functions in post-MI mice. Meanwhile, the genetic inhibition of PFKFB3 decreased the cardiac fibroblast activation and reversed the differentiated phenotypes in vitro and in vivo. Mechanistically, we identified deubiquitinase OTUD4 as a new binding protein of PFKFB3, and their interaction blocked PFKFB3 degradation via OTUD4-mediated deubiquitylation. Taken together, this work characterized a key role for PFKFB3 in cardiac fibroblast activation and suggested that inhibiting PFKFB3-involved glycolysis is an alternative way to alleviate post-MI cardiac fibrosis.Key messagesPFKFB3, a master activator of glycolysis, was highly expressed in ischemic cardiac fibroblasts to enhance cardiac fibrosisThe deubiquitinase OTUD4 was identified as a new binding protein of PFKFB3TGF-β1 blunted the ubiquitination-mediated degradation of PFKFB3 via OTUD4-mediated deubiquitylationBlockade of PFKFB3 contributed to ameliorating ischemia-induced cardiac fibrosisSupplementary InformationThe online version contains supplementary material available at 10.1007/s00109-023-02323-6.