Chronic kidney diseases (CKD) generally lead to renal fibrosis. Despite great progresses have been made in identifying molecular mediators of fibrosis, the mechanism that governs renal fibrosis remains unclear and so far no effective therapeutic anti-fibrosis strategy is available. Here we demonstrated that a switch of metabolism from oxidative phosphorylation to aerobic glycolysis (Warburg effect) in renal fibroblasts was the primary feature of fibroblast activation during renal fibrosis, and that suppressing renal fibroblast aerobic glycolysis could significantly reduce renal fibrosis. Both gene and protein assay showed that the expression of glycolysis enzymes were upregulated in mouse kidneys with unilateral ureter obstruction (UUO) surgery or in TGFβ1-treated renal interstitial fibroblasts. Aerobic glycolysis flux, indicated by glucose uptake and lactate production, was increased in mouse kidney with UUO nephropathy or TGFβ1-treated renal interstitial fibroblasts and positively correlated with fibrosis process. In line with this, we found that increasing aerobic glycolysis can remarkedly induce myofibroblasts activation while aerobic glycolysis inhibitors shikonin and 2-deoxyglucose (2DG) attenuate UUO-induced mouse renal fibrosis and TGFβ1-stimulated myofibroblast activation. Furthermore, mechanistic study indicated that shikonin inhibits renal aerobic glycolysis via reducing phosphorylation of pyruvate kinase type M2 (PKM2), a rate-limiting glycolytic enzyme associated with cell reliance on aerobic glycolysis. In conclusion, our findings demonstrate the critical role of aerobic glycolysis in renal fibrosis and provide the treatment with aerobic glycolysis inhibitors as a potential anti-fibrotic strategy.