Established Py-3Y1-S2 rat fibroblast cells were used to evaluate whether NaHCO3 or Na2CO3 influences glucose metabolism in vitro, because factors that contribute to metabolic pathways are much simpler to be evaluated in cultured cells than in whole animal bodies. The effects of the carbonates on glucose consumption decreased at high concentrations, >5 mg/ml for Na2CO3 and >7 mg/ml for NaHCO3, because of the increased pH of the culture medium. The effects of the carbonates on glucose consumption were additive with those of vanadium and concanavalin A. Streptozotocin, alloxan, and nicotinamide, which induced diabetes in animals, reduced glucose consumption by Py-3Y1-S2 cells, and the inhibitory effects of these reagents were abolished by both Na2CO3 and NaHCO3. Finally, the carbonates increased lactate production from glucose in the cells, followed by the acceleration of lactate secretion into the culture medium. The present study clarified that NaHCO3 and Na2CO3 directly regulated glucose metabolism.