The activity of microglia is fundamental for the regulation of numerous physiological processes including brain development, synaptic plasticity, and neurogenesis, and its deviation from homeostasis can lead to pathological conditions, including numerous neurodegenerative disorders. Carnosine is a naturally occurring molecule with well-characterized antioxidant and anti-inflammatory activities, able to modulate the response and polarization of immune cells and ameliorate their cellular energy metabolism. The better understanding of microglia characteristics under basal physiological conditions, as well as the possible modulation of the mechanisms related to its response to environmental challenges and/or pro-inflammatory/pro-oxidant stimuli, are of utmost importance for the development of therapeutic strategies. In the present study, we assessed the activity of carnosine on human HMC3 microglial cells, first investigating the effects of increasing concentrations of carnosine on cell viability. When used at a concentration of 20 mM, carnosine led to a decrease of cell viability, paralleled by gene expression increase and decrease, respectively, of interleukin 6 and heme oxygenase 1. When using the maximal non-toxic concentration (10 mM), carnosine decreased nitric oxide bioavailability, with no changes in the intracellular levels of superoxide ion. The characterization of energy metabolism of HMC3 microglial cells under basal conditions, never reported before, demonstrated that it is mainly based on mitochondrial oxidative metabolism, paralleled by a high rate of biosynthetic reactions. The exposure of HMC3 cells to carnosine seems to ameliorate microglia energy state, as indicated by the increase in the adenosine triphosphate/adenosine diphosphate (ATP/ADP) ratio and energy charge potential. The improvement of cell energy metabolism mediated by 10 mM carnosine could represent a useful protective weapon in the case of human microglia undergoing stressing conditions.