Podocyte injury is a hallmark of glomerular diseases; however, the underlying mechanisms remain unclear. B7-1 is increased in injured podocytes, but its intrinsic role is controversial. The clinical data here revealed the intimate correlation of urinary B7-1 with severity of glomerular injury. Through transcriptomic and biological assays in B7-1 transgenic and adriamycin nephropathy models, we identified B7-1 is a key mediator in podocyte injury and glomerulosclerosis through a series of signal transmission to β-catenin. Using LC-MS/MS, Hsp90ab1, a conserved molecular chaperone, was distinguished to be an anchor for transmitting signals from B7-1 to β-catenin. Molecular docking and subsequent mutant analysis further identified the residue K69 in the N terminal domain of Hsp90ab1 was the key binding site for B7-1 to activate LRP5/β-catenin pathway. The interaction and biological functions of B7-1-Hsp90ab1-LRP5 complex were further demonstrated in vitro and in vivo. We also found B7-1 is a novel downstream target of β-catenin. Our results indicate an intercrossed network of B7-1, which collectively induces podocyte injury and glomerulosclerosis. Our study provides an important clue to improve the therapeutic strategies to target B7-1.© 2022. The Author(s).