Advanced glycation end-products (AGEs) are extremely accumulated in the retinal vascular and epithelial cells of diabetes mellitus (DM) patients, particularly with diabetic retinopathy (DR). To elucidate the pathogenesis of the AGE-induced toxicity to retinal epithelial cells, we investigated the role of Fas-Fas ligand (FasL) signaling and mitochondrial dysfunction in the AGE-induced apoptosis. Results demonstrated that the AGE-BSA- induced apoptosis of retinal ARPE-19 cells. And the AGE-BSA treatment caused mitochondrial dysfunction, via deregulating the B-cell lymphoma 2 (Bcl-2) signaling. Moreover, the Fas/FasL and its downstreamer Caspase 8 were promoted by the AGE-BSA treatment, and the exogenous α-Fas exacerbated the activation of Caspase 3/8. On the other side, the siRNA-mediated knockdown of Fas/FasL inhibited the AGE-BSA-induced apoptosis. Taken together, we confirmed the activation of Fas-FasL signaling and of mitochondrial dysfunction in the AGE-BSA-promoted apoptosis in retinal ARPE-19 cells, implying the important role of Fas-FasL signaling in the DR in DM.