The aim of this study was to demonstrate the effects of the AMP-activated protein kinase (AMPK) activator 5-amino-4-imidazolecarboxamide riboside (AICAR) in combination with arsenic trioxide (ATO) in acute myeloid leukemia cells and determine its mechanism of action. Cell lines were either exposed to each drug alone or both the drugs simultaneously. Cell proliferation, cell cycle and apoptosis were assessed. Combination index (CI) method was used to calculate the synergistic, additive, or antagonistic effects of these drugs. Western blot technique was used to study the signaling molecules in the AMPK/TSC2/mTOR pathway. Simultaneous exposure of HL-60 cells to AICAR and ATO indicated a synergism (CI < 1), whereas CI on NB4 cells was greater than 1. In HL-60, the change in expression level of each protein was quite significant in the presence of the combination as compared to that induced through any single agent. On the contrary, ATO weakened the effect of AICAR-mediated AMPK activation in NB4 cells. ATO caused a profound decrease in the protein level of PML/RARalpha in NB4 cells after 48 h, but there was no change with AICAR and the combination. The combination of AICAR and ATO produced a synergistic effect in the treatment of HL-60 cells involving AMPK/TSC2/mTOR pathway, and AICAR reduced ATO-mediated apoptotic death on acute promyelocytic leukemia NB4 cells.