Excess reactive oxygen species (ROS) production is thought to play a key role in the loss of pancreatic β-cell number and/or function, in response to high glucose and/or fatty acids. However, contradictory findings have been reported showing that in pancreatic β cells or insulin-secreting cell lines, ROS are produced under conditions of either high or low glucose. Superoxide production was measured in attached INS1E cells as a function of glucose concentration, by following in real time the oxidation of dihydroethidine. Minimal values of superoxide production were measured at glucose concentrations of 5–20mM, whereas superoxide generation was maximal at 0–1mM glucose. Superoxide generation started rapidly (15–30min) after exposure to low glucose and was suppressed by its addition within minutes. Superoxide was totally suppressed by rotenone, but not myxothiazol, suggesting a role for complex I in this process. Indirect evidence for mitochondrial ROS generation was also provided by a decrease in aconitase activity. Activation of AMPK, a cellular metabolic sensor, and its downstream target ACC by low glucose concentration was largely inhibited by addition of MnTBAP, a MnSOD and catalase mimetic that also totally suppressed superoxide production. Taken together, the data show that low glucose activates AMPK in a superoxide-dependent, AMP-independent way.