Evidence of diabetes-induced nuclear factor-κB (NF-κB) activation has been provided with DNA binding assays or nuclear localization with immunohistochemistry, but few studies have explored mechanisms involved. We examined effects of diabetes on proteins comprising NF-κB canonical and noncanonical activation pathways in the renal cortex of diabetic mice. Plasma concentrations of NF-κB–regulated cytokines were increased after 1 month of hyperglycemia, but most returned to control levels or lower by 3 months, when the same cytokines were increased significantly in renal cortex. Cytosolic content of NF-κB canonical pathway proteins did not differ between experimental groups after 3 months of diabetes, while NF-κB noncanonical pathway proteins were affected, including increased phosphorylation of inhibitor of κB kinase-α and several fold increases in NF-κB–inducing kinase and RelB, which were predominantly located in tubular epithelial cells. Nuclear content of all NF-κB pathway proteins was decreased by diabetes, with the largest change in RelB and p50 (approximately twofold decrease). Despite this decrease, measurable increases in protein binding to DNA in diabetic versus control nuclear extracts were observed with electrophoretic mobility shift assay. These results provide evidence for chronic NF-κB activation in the renal cortex of db/db mice and suggest a novel, diabetes-linked mechanism involving both canonical and noncanonical NF-κB pathway proteins.