AMP-activated protein kinase (AMPK) is being primarily studied as a central metabolic stress sensor, which regulates cell survival and growth-related metabolic pathways to preserve intracellular ATP levels in response to energy deprivation. Evidence is now accumulating that AMPK plays also an obligatory role to ensure proper cell division and faithful chromosomal segregation during mitosis. Increased phosphorylation in the AMPK catalytic subunit (AMPKα) was found in a proteomic study for kinases activated during G2/M and, more recently, activated AMPKα (PP-AMPKα(Thr172)) has been observed to transiently associate with several mitotic structures including centrosomes, spindle poles, the central spindle midzone and the midbody throughout all of the mitotic stages and cytokinesis. How AMPKα activation is controlled spatially and temporally during mitosis, however, remained undiscovered. Because Polo-like Kinases (PLKs) regulate many aspects of mitotic progression including centrosome maturation, bipolar spindle assembly, chromosome congression & segregation, and cytokinesis, we decided to combine an immunofluorescence microscopy analysis with a chemical biology approach employing a small-molecule PLK1 inhibitor to dissect a putative relationship between PLK1 and AMPKα during G2/M transition. PLK1 and PP-AMPKα(Thr172) were found to display a major spatio-temporal overlap early at centrosomes, from prophase until anaphase, and late at the midbody, during telophase and cytokinesis. Moreover, short-term treatment with the thiophene derivative GW843682X, a selective PLK inhibitor that has 400-fold greater potency for PLK1 than for PLK2 or PLK3, fully abolished mitotic activation of AMPKα. Upon long-term PLK1 inhibition, PP-AMPKα(Thr172) was barely detected surrounding the spindle poles of prometaphase-like arrested cells displaying the "polo" phenotype. Similarly, PP-AMPKα(Thr172) was largely inhibited in GW843682X-treated cells exhibiting cytokinesis failure and binucleate cell formation. Given that PLK1 is a well-recognized master regulatory kinase for the numerous protein substrates involved in mitosis, our current description of a causal link between PLK1 activity and mitotic phosphorylation of AMPKα may provide fundamental insights into how the energy sensor AMPK is directly coupled to mitotic cell division and cell cycle exist.