Microglia activation is a metabolically demanding process that implies the transition to a pro- or anti-inflammatory phenotype. During inflammation, microglia undergo metabolic reprogramming to meet their energy needs. Thus, the pro-inflammatory phenotype derives its energy mainly from aerobic glycolysis, whereas the anti-inflammatory phenotype depends on oxidative phosphorylation. The modulation of metabolic pathways in activated microglia could change their phenotype. Exogenously administered agmatine pretreatment (Agm, 100 µM) modulates the Lps-activated BV-2 microglial cell line towards an anti-inflammatory phenotype. We used the same experimental setup to investigate whether agmatine affects glycolysis. Using immunofluorescence microscopy, we analysed the transcription factor hypoxia-inducible factor-1α (HIF-1α), which is known to regulate glycolysis in microglia under inflammatory conditions. Using qPCR, we analysed the expressions of HIF-1α-responsive genes involved in the regulation of glycolysis: Slc2a1, encoding the glucose transporter GLUT-1, and Pfkfb3, encoding 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3, a key enzyme involved in increasing glycolytic flux. In addition, lactate production was assessed and served as a measure of the glycolytic rate. In contrast to the low HIF-1α signal in unstimulated cells, Lps activation of microglia resulted in marked HIF-1α nuclear positivity, which was reduced by Agm pretreatment. Lps increased the expression of Slc2a1 and Pfkfb3, while Agm decreased it significantly in stimulated cells. As a result of the change in Slc2a1 expression, Agm reduced GLUT-1 protein levels upregulated by Lps. In addition, Agm partially prevented lactate production promoted by Lps. These results suggest the ability of Agm to attenuate glycolysis in pro-inflammatory microglia via the HIF-1α signalling pathway.