Advanced glycation end-products (AGEs), epidermal growth factor receptor (EGFR), reactive oxygen species (ROS), and extracellular signal-regulated kinases (ERK) are implicated in diabetic nephropathy (DN). Therefore, we asked if AGEs-induced ERK protein phosphorylation and mitogenesis are dependent on the receptor for AGEs (RAGE)–ROS–EGFR pathway in normal rat kidney interstitial fibroblast (NRK-49F) cells. We found that AGEs (100 µg/ml) activated EGFR and ERK1/2, which was attenuated by RAGE short-hairpin RNA (shRNA). AGEs also increased RAGE protein and intracellular ROS levels while RAGE shRNA and N-acetylcysteine (NAC) attenuated AGEs-induced intracellular ROS. Hydrogen peroxide (5–25 µM) increased RAGE protein level while activating both EGFR and ERK1/2. Low-dose hydrogen peroxide (5 µM) increased whereas high-dose hydrogen peroxide (100 µM) decreased mitogenesis at 3 days. AGEs-activated EGFR and ERK1/2 were attenuated by an anti-oxidant (NAC) and an EGFR inhibitor (Iressa). Moreover, AGEs-induced mitogenesis was attenuated by RAGE shRNA, NAC, Iressa, and an ERK1/2 inhibitor (PD98059). In conclusion, it was found that AGEs-induced mitogenesis is dependent on the RAGE–ROS–EGFR–ERK1/2 pathway whereas AGEs-activated ERK1/2 is dependent on the RAGE–ROS–EGFR pathway in NRK-49F cells. J. Cell. Biochem. 109: 38–48, 2010. © 2009 Wiley-Liss, Inc.