AMP-activated protein kinase (AMPK) is an energy-sensing enzyme that is activated by an increased AMP/ATP ratio. AMPK is now well-recognized to induce glucose uptake in skeletal muscle and heart. AICAR is phosphorylated to form the AMP analogue, ZMP, which activates AMPK. Its effects on glucose transport appear to be tissue-specific. The purpose of our study was to examine the effect of AICAR on insulin-induced glucose uptake in adult rat ventricular cardiomyocytes. We studied isolated adult rat ventricular cardiomyocytes treated or not with the AMPK activators, AICAR and metformin, and subsequently, with insulin or not. Insulin action was investigated by determining deoxyglucose uptake, IRS-1- or IRS-2-associated PI3K activity and PKB cascade using antibodies to PKB, GSK3, and AS160. Intracellular pH was evaluated using the fluorescent pH-sensitive dye BCECF, and NHE1 activity was assessed using the NH₄⁺ prepulse method. Our key findings are as follows. AICAR and metformin enhance insulin signaling downstream of PKB. Metformin potentiates insulin-induced glucose uptake, but, surprisingly, AICAR inhibits both basal and insulin-induced glucose uptake. Moreover, we found that AICAR decreases intracellular pH, via inhibition of the Na⁺/H⁺ exchanger NHE1. In conclusion, AMPK potentiates insulin signaling downstream of PKB in isolated cardiac myocytes, consistent with findings in the heart in vivo. Further, AICAR inhibits basal and insulin-induced glucose uptake in isolated cardiac myocytes via the inhibition of NHE1 and the subsequent reduction of intracellular pH. Importantly, AICAR exerts these effects in a manner independent of AMPK activation.