Glucose is the preferred carbon source for most cell types and a major determinant of cell growth. In yeast and certain mammalian cells, glucose activates the cAMP‐dependent protein kinase A (PKA), but the mechanisms of PKA activation remain unknown. Here, we identify cytosolic pH as a second messenger for glucose that mediates activation of the PKA pathway in yeast. We find that cytosolic pH is rapidly and reversibly regulated by glucose metabolism and identify the vacuolar ATPase (V‐ATPase), a proton pump required for the acidification of vacuoles, as a sensor of cytosolic pH. V‐ATPase assembly is regulated by cytosolic pH and is required for full activation of the PKA pathway in response to glucose, suggesting that it mediates, at least in part, the pH signal to PKA. Finally, V‐ATPase is also regulated by glucose in the Min6 β‐cell line and contributes to PKA activation and insulin secretion. Thus, these data suggest a novel and potentially conserved glucose‐sensing pathway and identify a mechanism how cytosolic pH can act as a signal to promote cell growth.