Podocalyxin represents a Wilms’ tumor suppressor protein (WT1)-regulated differentiation marker for glomerular epithelium. We provide evidence concerning mechanisms involved in the regulation of podocalyxin expression following long-term exposure to increased (25 mM) glucose levels. Prolonged culture of conditionally immortalized human podocytes in 25 mM glucose induced suppression of podocalyxin expression both at the protein and mRNA levels, whereas WT1 protein levels remained unaltered. WT1 interacted with another transcription factor, CRE-binding protein (CBP). This association was decreased by 40% in the presence of 25 mM glucose. Chromatin immunoprecipitation assays on chromatin from podocytes cultured in 25 mM glucose revealed reduced WT1 binding to podocalyxin promoter sequences, probably resulting from impaired WT1-CBP interactions. We explored the possible role of glucose-induced adducts (advanced glycation end products; AGEs) in impairing interactions between WT1 and CBP, with the use of aminoguanindine, an inhibitor of AGE formation. Podocytes were cultured in the simultaneous presence of 20 mM aminoguanidine and 25 mM glucose, and podocalyxin protein levels were examined. Aminoguanidine effectively prevented downregulation of podocalyxin protein levels but could not restore podocalyxin levels once expression was suppressed. Thus increased glucose apparently impaired the ability of WT1 to initiate transcription in part by decreased association of WT1 with CBP. Administration of aminoguanidine concomitant with increasing glucose levels in our in vitro model system protected from glucose-induced “silencing” of the podocalyxin gene, suggesting that AGEs play an important role in suppressing its expression in diabetic conditions.