Advanced glycation end products (AGEs) play a role in pathogenesis of diabetic nephropathy (DN). Myo-inositol oxygenase (MIOX) has been implicated in tubulointerstitial injury in the context of DN. We investigated the effect of AGEs on MIOX expression and delineated mechanisms that lead to tubulointerstitial injury. The status of MIOX, RAGE, and relevant cellular signaling pathways activated following AGE:RAGE interaction was examined in tubular cells and kidneys of AGE-BSA-treated mice. A solid-phase assay revealed an enhanced binding of RAGE with AGE-BSA, AGE-laminin, and AGE-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 phosphatidylinositol 3-kinase (PI3K)-AKT pathway and increased expression of NF-κB, transforming growth factor (TGF)-β, and fibronectin, which was negated with the treatment of MIOX/RAGE- small interfering (si) RNA. Concomitant with MIOX upregulation, 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, upregulation of MIOX, RAGE and NF-κB, along with influx of monocytes into the tubulointerstitium, increased the expression of MCP-1, IL-6, and fibronectin and increased the 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 upregulation of MIOX, with excessive generation of ROS, increased expression of NF-κB, inflammatory cytokines, TGF-β, and fibronectin. Collectively, these observations highlight the relevance of the biology of MIOX in the contribution toward tubulointerstitial injury in DN.