OBJECTIVE Chronic kidney disease (CKD) in diabetes has a complex molecular and likely multifaceted pathophysiology. We aimed to validate a panel of biomarkers identified using a systems biology approach to predict the individual decline of estimated glomerular filtration rate (eGFR) in a large group of patients with type 2 diabetes and CKD at various stages. RESEARCH DESIGN AND METHODS We used publicly available “omics” data to develop a molecular process model of CKD in diabetes and identified a representative parsimonious set of nine molecular biomarkers: chitinase 3-like protein 1, growth hormone 1, hepatocyte growth factor, matrix metalloproteinase (MMP) 2, MMP7, MMP8, MMP13, tyrosine kinase, and tumor necrosis factor receptor-1. These biomarkers were measured in baseline serum samples from 1,765 patients recruited into two large clinical trials. eGFR decline was predicted based on molecular markers, clinical risk factors (including baseline eGFR and albuminuria), and both combined, and these predictions were evaluated using mixed linear regression models for longitudinal data. RESULTS The variability of annual eGFR loss explained by the biomarkers, indicated by the adjusted R2 value, was 15% and 34% for patients with eGFR ≥60 and <60 mL/min/1.73 m2, respectively; variability explained by clinical predictors was 20% and 31%, respectively. A combination of molecular and clinical predictors increased the adjusted R2 to 35% and 64%, respectively. Calibration analysis of marker models showed significant (all P < 0.0001) but largely irrelevant deviations from optimal calibration (calibration-in-the-large: −1.125 and 0.95; calibration slopes: 1.07 and 1.13 in the two groups, respectively). CONCLUSIONS A small set of serum protein biomarkers identified using a systems biology approach, combined with clinical variables, enhances the prediction of renal function loss over a wide range of baseline eGFR values in patients with type 2 diabetes and CKD.