<p>Diabetic nephropathy (DN) is one of the most serious and common diabetes-associated complications. Licochalcone B is a chalcone that has been found to possess anti-diabetic activity. However, protective potential of licochalcone B in DN remains unclear. In this study, we investigated the protective effect of licochalcone B against DN both in vitro and in vivo. The results showed that licochalcone B protected HK-2 cells from high glucose (HG)-induced oxidative stress and ferroptosis. Further molecular docking and molecular dynamics simulation revealed the stable binding abilities of licochalcone B and activating transcription factor 3 (ATF3). We found that licochalcone B negatively regulated ATF3 expression. Licochalcone B also inhibited the HG-induced ATF3 expression in HK-2 cells. ATF3 overexpression reversed the inhibitory effects of licochalcone B on oxidative stress and ferroptosis in HG-induced HK-2 cells. Moreover, ATF3 overexpression downregulated the expression levels of solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4), which are two critical regulators of ferroptosis. In vivo assays also proved that licochalcone B administration significantly improved histopathological changes in kidney tissues from HFD/STZ mice. Overall, our findings suggested that licochalcone B exerted protective effect against DN through ameliorating oxidative stress and ferroptosis via regulating ATF3/SLC7A11/GPX4 axis. These results highlighted the therapeutic potential of licochalcone B in preventing and treating DN.</p>