Recent studies have reported that high glucose (HG) conditions may contribute to the acceleration of renal cell apoptosis and renal fibrosis by inducing epithelial-mesenchymal transition (EMT) of tubular epithelial cells, in which c-Src kinase and transforming growth factor (TGF)-β are key modulators. In the present study, the roles of c-Src kinase and TGF-β in EMT of lens epithelial cells (LECs) under HG conditions were investigated. Results indicated human lens epithelial B3 (HLE-B3) cells under HG conditions exhibited significantly increased protein expression levels of phosphorylated c-Src (p-Src) (P<0.05) and secreted a significantly increased amount of TGF-β compared with HLE-B3 cells under normal glucose conditions (P<0.05). Notably the c-Src inhibitor PP1 and the activin receptor-like kinase 5 (ALK5) inhibitor SB431542 suppressed EMT of HLE-B3 cells. Results indicated that PP1 significantly inhibited the activities of c-Src and ALK5 and the secretion of TGF-β, whereas SB431542 only significantly downregulated the protein expression levels and secretion of TGF-β (P<0.05). Following c-Src knockdown, the protein expression levels of p-Src, ALK5 and TGF-β were significantly decreased, the secretion of TGF-β was significantly suppressed (both P<0.05) and EMT was decreased in HLE-B3 cells. These results suggest that c-Src and TGF-β may promote EMT of LECs under HG conditions, with c-Src as the upstream regulatory molecule. Thus, the signal axis of c-Src/TGF-β in EMT of LECs may be a potential novel therapeutic target for the prevention of diabetic subcapsular cataract.