AMP-dependent kinase (AMPK) agonists, the biguanide metformin and 5-aminoimidazole-4-carboxamide ribonucleoside (AICAr) inhibit proliferation of leukemia cell lines, but only AICAr induces expression of differentiation markers in monocytic U937 cell line. The mechanism of AICAr-induced differentiation is not fully understood, but it is known to be AMPK-independent. In this study, we investigated the role of glucose and glutamine metabolism in AICAr-mediated effects in leukemia cell lines. We used commercially available kits for measuring glucose, lactate and ammonia in response to AMPK-agonists and all-trans-retinoic acid (ATRA) as an inducer of granulocytic differentiation. The results showed that ATRA decreased, metformin increased, and AICAr had no effects on glucose consumption and lactate production. However, AICAr increased ammonia production, and the increase was abolished when U937 cells were grown in the absence of glutamine or in the presence of bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)-ethyl sulphide (BPTES), a pharmacological inhibitor of glutaminase 1 (GLS1). Flow cytometry analyses revealed that the lack of glutamine and the inhibition of GLS1 inhibited the expression of differentiation markers in response to AICAr, but had no effects on ATRA-mediated differentiation. These results show that glutamine metabolism is associated with AICAr-mediated differentiation of U937 cells.