Burgeoning evidence points to glycogen synthase kinase (GSK)3β as a key player in diverse kidney diseases. However, as a pivotal transducer of the insulin signaling pathway, the role of GSK3β in diabetic kidney disease remains uncertain. In db/db mice, renal expression of total and activated GSK3β was increasingly elevated. This preceded the development of diabetic kidney disease, and correlated with the progression of signs of diabetic kidney injury, including albuminuria and extracellular matrix accumulation in glomeruli and tubulointerstitia. In vitro, exposure of glomerular podocytes, mesangial cells, and renal tubular cells to a diabetic milieu induced GSK3β overexpression and hyperactivity, which seem essential and sufficient for eliciting diabetic cellular damages in kidney cells, because the cytopathic effect of the diabetic milieu was mitigated by GSK3β knockdown, but was mimicked by ectopic expression of constitutively active GSK3β even in the normal milieu. In consistency, kidney biopsy specimens procured from patients with varying stages of diabetic nephropathy revealed an amplified expression of total and activated GSK3β in glomeruli and renal tubules, associated with the severity of diabetic nephropathy. Moreover, in retrospective cohorts of type 2 diabetic patients that were followed for over five years, the relative activity of GSK3β in banked urinary exfoliated cells represented an independent risk factor for development or progression of renal impairment. Furthermore, receiver operating characteristic curve analysis demonstrated that GSK3β activity in urinary exfoliated cells provided much better power than albuminuria in discriminating diabetic patients with progressive renal impairment from those with stable kidney function. Thus, renal expression and activity of GSK3β are amplified in experimental and clinical diabetic nephropathy. Hence, GSK3β in urinary exfoliated cells may serve as a novel biomarker for predicting diabetic kidney disease progression.