Acute kidney injury (AKI) is a highly prevalent medical syndrome associated with high mortality and morbidity. Several types of cells, including epithelial cells, vascular endothelial cells, pericytes, and macrophages, participate in the development of AKI. Recently, renal fibroblasts were found to play an important role in the regulation of tubular injury, repair, and recovery after AKI. However, the mechanisms underlying fibroblast activation and proliferation during the progression of AKI remain unclear. In the present study, we found many activated myofibroblasts located in the renal interstitium with an abundance of extracellular matrix deposition following folic acid-induced AKI. The proliferative pattern of tubular epithelial cells and interstitial cells following acute injury was different, indicating that the proliferation of fibroblasts followed the proliferation of tubular epithelial cells. Furthermore, we observed that proliferative tubular epithelial cells preferred aerobic glycolysis as the dominating metabolic pathway in the progression of AKI. Lactate generated from injured tubules was taken up by interstitial fibroblasts in the later stages of AKI, which induced fibroblast activation and proliferation in vitro. Early inhibition of lactate production in tubules by glycolytic inhibitors suppressed fibroblast activation after folic acid-induced injury. Collectively, these results support the important role of fibroblasts in the development of AKI and suggest that lactate produced by glycolysis in tubular epithelial cells is a novel regulator of fibroblast activation and proliferation.