Diabetic nephropathy (DN) is associated with renal interstitial injury and fibrosis. Our previous study showed that andrographolide protected against the progression of DN and high glucose (HG)-induced mesangial dysfunction. However, the protective effects of andrographolide on renal tubular epithelial cells have not been fully elucidated.To determine the protective effects of andrographolide on renal tubular damage and explore the underlying mechanism.Human tubular epithelial cells (HK-2 cells) were treated with andrographolide (5 and 10 μM) under HG conditions. Diabetic mice were treated with andrographolide (i.p. 2 and 4 mg/kg, twice per week).Western blotting, reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence and flow cytometry were used to analyze the effects of andrographolide on renal tubular injury and fibrotic mechanisms in HK-2 cells. The protective effects of andrographolide against renal tubulointerstitial injury and fibrosis were investigated in diabetic mice fed a high-fat diet (HFD). Renal interstitial tissue was collected at sacrifice for immunohistochemistry, immunofluorescence analysis, RT-PCR and Western blotting to analyze the effects of andrographolide on renal tubular injury and fibrosis.In vitro assay results indicated that andrographolide (5 and 10 μM) effectively inhibited HG-induced apoptosis, epithelial-mesenchymal transition (EMT) and collagen deposition in HK-2 cells. Mechanistically, HG stimulated mitochondrial reactive oxygen species (mtROS)-mediated NOD-like receptor family and pyrin domain-containing protein 3 (NLRP3) inflammasome activation and EMT in tubular epithelial cells, and andrographolide (5 and 10 μM) inhibited these effects by ameliorating mitochondrial dysfunction. In vivo, treatment with andrographolide (2 and 4 mg/kg) inhibited renal tubular cell apoptosis, EMT and tubulointerstitial fibrosis, mitochondrial dysfunction and NLRP3 inflammasome activation in diabetic mice.Andrographolide (5 and 10 μM) prevents HG-induced renal tubular cell damage, and andrographolide (2 and 4 mg/kg) protects against the progression of diabetic tubular injury and fibrosis in mice by suppressing mitochondrial dysfunction and NLRP3 inflammasome activation.Copyright © 2021. Published by Elsevier GmbH.