Dysmetabolic state in diabetes leads to augmented synthesis of extracellular matrix(ECM) proteins. In the endothelial cells, we have previously demonstrated that glucose-induced fibronectin(FN) and its splice variant EDB⁺FN production are regulated by protein kinase B(PKB, a.k.a Akt). Here we investigated role of Akt1 in ECM protein production in the organs affected by chronic diabetic complications. We studied Akt1/PKB knockout mice and wild-type controls. To avoid confounding effects of systemic insulin, we used 30% galactose feeding to induce hyperhexosemia for 8 weeks starting at 6 weeks of age. We investigated FN mRNA, EDB⁺FN mRNA, and transforming growth factor(TGF) mRNA expression, Akt phosphorylation, Akt kinase activity, and NFB and AP-1 activation in the retina, heart and kidney. Renal and cardiac tissues were histologically examined. Galactose feeding caused significant upregulation of FN, EDB⁺FN and TGF- in all tissues. FN protein levels paralleled mRNA. Such upregulation were prevented in Akt1-deficient galactose-fed mice. Galactose feeding caused ECM protein deposition in the glomeruli and in the myocardium, which were prevented in the Akt-knockout mice. NFB and and AP-1 activation was pronounced in galactose-fed wild-type mice and were prevented in galactose-fed Akt1/PKB deficient group. In the retina and kidney, serine 473 was predominant site for Akt phosphorylation whereas in the heart it was threonine 308. Parallel experiment in streptozotocin-induced diabetic animals showed similar results. The data from this study indicate that hyperhexosemia-induced Akt/PKB activation is an important mechanism leading to NF-B and AP-1 activation and increased ECM protein synthesis in the organs affected by chronic diabetic complications.