Our previous studies have suggested a critical role of reticulon (RTN)1A in mediating endoplasmic reticulum (ER) stress in kidney cells of animal models and humans with kidney diseases. A large body of evidence suggests that proteinuria itself can cause tubular cell injury leading to the progression of kidney disease. In the present study, we determined whether RTN1A mediates proteinuria-induced tubular cell injury through increased ER stress. We found that incubation of HK2 cells with human serum albumin induced the expression of RTN1A and ER stress markers, whereas knockdown of RTN1A expression attenuated human serum albumin-induced ER stress and tubular cell apoptosis in vitro. In vivo, we found that tubular cell-specific RTN1 knockdown resulted in a significant attenuation of tubular cell ER stress, apoptosis, and renal fibrosis in a model of albumin overload nephropathy. Based on these findings, we conclude that RTN1A is a key mediator for proteinuria-induced tubular cell toxicity and renal fibrosis.