Mitochondria are extremely dynamic organelles, constantly undergoing antagonistic processes of fission and fusion. Multiple mitochondrial membrane GTPases regulate mitochondrial dynamics. Among them, Dynamin-related protein 1 (Drp1) executes fission by recruiting to the mitochondria outer membrane to drive scission. Drp1 activity can be reversibly modified by two critical phosphorylation sites. Phosphorylation of Drp1 at serine 616 (p-Drp1S616) promotes Drp1 activity. Conversely, phosphorylation of serine 637 (p-Drp1S637) represses its activity. Besides controlling morphology, mitochondrial dynamics play crucial roles in bioenergetics, metabolism, cell transformation, proliferation, and apoptosis. The imbalance of fission and fusion dynamics resulting in excessive mitochondrial fragmentation has been implicated in many chronic illnesses. Excessive mitochondrial fission acts as a pro-proliferative marker in lung fibrosis, its potential role in renal fibroblast activation and fibrogenesis has never been investigated. Mitochondrial dynamic features of fibroblasts were examined in fibrotic and nonfibrotic kidney. The potential role of Drp1-mediated mitochondrial fission in renal fibroblasts activation, proliferation and fibrogenesis using in vivo, ex vivo, and in vitro strategies. We showed more pronounced fragmented mitochondria in fibrotic than in non-fibrotic renal fibroblasts in humans(Figure 1A) and mice(Figure 1B), along with the increased phosphorylation of Drp1 at serine 616. Pharmacological inhibition of mitochondrial fission by mdivi-1(a specific inhibitor of Mdivi-1) attenuated myofibroblast accumulation and interstitial fibrosis in the obstructive kidneys of mice(Figure 1C and 1D). Moreover, ex vivo mdivi-1 treatment reversed mitochondrial fission and differentiated phenotype in primary myofibroblast-derived from fibrotic kidney. In vitro study, by using a rat fibroblast cell line(NRK-49F), Drp1 knockdown by using siRNA suppressed TGF-β1-elicited mitochondrial fragmentation, mtROS elevation and glycolytic shift, which promoted apoptosis and inhibited myofibroblast differentiation and proliferation in NRK-49F cells(Figure 1E and 1F). Further, phosphorylation of Drp1 at serine 616 (p-Drp1S616) and acetylation of H3K27(H3K27ac) was upregulated in fibrotic kidneys and TGF-β1-treated NRK-49F cells. Overexpression of inactive mutant of Drp1(Drp1S616A) rather than wild-type Drp1 rescued TGF-β1-induced myofibroblastic phenotype, along with reduced p-Drp1S616 and H3K27ac in Drp1-deleted NRK-49F cells. TGF-β1 treatment increased the enrichment of H3K27ac at the promoters of α-SMA and PCNA, which was hampered by overexpressed Drp1S616A but not wild-type Drp1 in Drp1-deficient cells.(For the reason that only one images can be uploaded, the rest of the data are not shown in the figure) Our results imply that mitochondrial dynamics is crucial for fibroblasts activation and proliferation, modulating Drp1-mediated mitochondrial fission in fibroblast may serve as a therapeutic target for retarding renal fibrogenesis.