As an endo-β (1-4)-D: -glucuronidase, heparanase can specifically cleave carbohydrate chains of heparan sulfate (HS) and has been implicated in development of endothelial cells dsyfunction. The advanced glycation end products (AGEs) play a pivotal role in the pathology of diabetic complications. In the present study, we investigated the effect of AGE-bovine serum albumin (AGE-BSA) on heparanase expression in human microvascular endothelial cells (HMVECs) and the underlying molecular mechanisms. The results indicated that in vitro direct exposure of HMVECs to AGE-BSA (300, 1000, and 3000 μg/ml) could increase heparanase mRNA and protein expression in a dose and time-dependent manner. The effect of 1000 μg/ml AGE-BSA could be abolished by neutralization with antibody of the receptor for advanced glycation end products (RAGE). Moreover, pretreatment with inhibitors of nuclear factor-κB (NF-κB) or PI3-kinase did not affect heparanase expression induced by AGE-BSA. Nevertheless, small interference RNA (siRNA) for transcriptional factor FOXO4 could reduce the increase of heparanase expression in HMVECs induced by 1000 μg/ml AGE-BSA. These results suggest that AGEs could induce heparanase expression in HMVECs by RAGE and predominantly through activation of the FOXO4 transcription factor.