Previously, our lab showed that the endoplasmic reticulum (ER) and calcium regulatory protein, calreticulin (CRT), is important for collagen transcription, secretion, and assembly into the extracellular matrix (ECM) and that ER CRT is critical for TGF-β stimulation of type I collagen transcription through stimulation of ER calcium release and NFAT activation. Diabetes is the leading cause of end stage renal disease. TGF-β is a key factor in the pathogenesis of diabetic nephropathy. However, the role of calreticulin (Calr) in fibrosis of diabetic nephropathy has not been investigated. In current work, we used both in vitro and in vivo approaches to assess the role of ER CRT in TGF-β and glucose stimulated ECM production by renal tubule cells and in diabetic mice. Knockdown of CALR by siRNA in a human proximal tubular cell line (HK−2) showed reduced induction of soluble collagen when stimulated by TGF-β or high glucose as compared to control cells, as well as a reduction in fibronectin and collagen IV transcript levels. CRT protein is increased in kidneys of mice made diabetic with streptozotocin and subjected to uninephrectomy to accelerate renal tubular injury as compared to controls. We used renal-targeted ultrasound delivery of Cre-recombinase plasmid to knockdown specifically CRT expression in the remaining kidney of uninephrectomized Calrfl/fl mice with streptozotocin-induced diabetes. This approach reduced CRT expression in the kidney, primarily in the tubular epithelium, by 30–55%, which persisted over the course of the studies. Renal function as measured by the urinary albumin/creatinine ratio was improved in the mice with knockdown of CRT as compared to diabetic mice injected with saline or subjected to ultrasound and injected with control GFP plasmid. PAS staining of kidneys and immunohistochemical analyses of collagen types I and IV show reduced glomerular and tubulointerstitial fibrosis. Renal sections from diabetic mice with CRT knockdown showed reduced nuclear NFAT in renal tubules and treatment of diabetic mice with 11R-VIVIT, an NFAT inhibitor, reduced proteinuria and renal fibrosis. These studies identify ER CRT as an important regulator of TGF-β stimulated ECM production in the diabetic kidney, potentially through regulation of NFAT-dependent ECM transcription.