Advanced glycation end products (AGEs) play a role in pathogenesis of diabetic nephropathy (DN). Myo-inositol Oxygenase (MIOX) has been implicated in tubulo-interstitial injury in the context of DN. We investigated the effect of AGEs on MIOX expression and delineated mechanisms that lead to tubulo-interstitial injury. Status of MIOX, RAGE and relevant cellular signaling pathways activated following AGE:RAGE interaction were examined in tubular cells and kidneys of AGE-BSA treated mice. Solid phase assay revealed an enhanced binding of RAGE with AGE-BSA, -laminin and -collagen IV. The cells treated with AGE-BSA had increased MIOX activity/expression and promoter activity. This was associated with activation of various signaling kinases of PI3K-AKT pathway, and increased expression of NF-B, TGF-β and fibronectin, which was negated with the treatment of MIOX/RAGE-siRNA. Concomitant with MIOX up-regulation there was an increased generation of reactive oxygen species (ROS), which could be abrogated with MIOX/RAGE-siRNA treatment. The kidneys of mice treated with AGE-BSA had significantly high urinary A/C ratio, up-regulation of MIOX, RAGE and NF-B along with influx of monocytes into the tubulo-interstitium, increased expression of MCP-1, IL-6 and fibronectin, and generation of ROS. Such perturbations were abrogated with the concomitant treatment of inhibitors MIOX or RAGE (D-glucarate and FPS-ZM1). These studies support a role of AGE:RAGE interaction in the activation of PI3K-AKT pathway and up-regulation of MIOX, with excessive generation of ROS, increased expression of NF-B, inflammatory cytokines, TGF-β and fibronectin. Collectively, these observations highlight the relevance of the biology of MIOX in the contribution towards tubulo-interstitial injury in DN.