Methylglyoxal (MGO), a reactive α-oxoaldehyde produced by glucose metabolism, is elevated in several diabetic complications, including diabetic retinopathy. The breakdown of retinal pigment epithelial cells is implicated in the progression of diabetic retinopathy. Increased concentrations of MGO lead to retinal pigment epithelial cell death. In this study, we investigated the involvement of activating transcription factor 6 (ATF6) in MGO-mediated cytotoxicity in ARPE-19 cells. In response to high concentrations of MGO, unfolded protein response-related ATF6 was induced. Interestingly, the MGO also induced the generation of reactive oxygen species (ROS) and the phosphorylation of ERK and p38 MAPK. The induction of ATF6 was inhibited by ERK-specific and p38 MAPK-specific inhibitors (U0126 and SB202190) and by NAC, a well-known ROS scavenger. NAC also attenuated the phosphorylation of p38 MAPK. MGO induced cytotoxicity in the ARPE-19 cells, which was ameliorated by the inhibition of the ERK, p38 MAPK, and ROS pathways. Furthermore, the MGO-mediated cytotoxicity was inhibited by ATF6 siRNA. Taken together, these results clearly show that the induction of ATF6 via the ERK and ROS-p38 MAPK pathways is implicated in MGO-induced cytotoxicity in ARPE-19 cells.