Streptococcus thermophilus is widely used in the dairy industry for the manufacturing of fermented milk and cheeses and probiotic formulations. S. thermophilus evolved from closely phylogenetically related pathogenic streptococci through loss-of-function events counterbalanced by the acquisition of relevant traits, such as lactose and urea utilization for the adaptation to the milk environment. In the context of regressive evolution, the urease gene cluster accounts for 0.9% of the total coding sequence belonging to known functional categories. The fate of ammonia and carbon dioxide derived by urea hydrolysis in several biosynthetic pathways have been depicted, and the positive effect of urease activity on S. thermophilus growth fitness and lactic acid fermentation in milk has been already addressed by several authors. However, the mechanistic effect of urea hydrolysis on the energetic metabolisms of S. thermophilus is still unclear. This study aimed to assess the effect of urease activity on the growth and energy metabolism of Streptococcus thermophilus in milk. In milk, 13C-urea was completely hydrolyzed in the first 150 min of S. thermophilus growth, and urea hydrolysis was accompanied by an increase in cell density and a reduction in the generation time. By using energetically discharged cells with gene transcription and translation blocked, we showed that in the presence of fermentable carbon sources, urease activity, specifically the production of ammonia, could dramatically boost glycolysis and, in cascade, homolactic fermentation. Furthermore, we showed that ammonia, specifically ammonium ions, were potent effectors of phosphofructokinase, a key glycolytic enzyme. IMPORTANCE Finding that ammonia-generating enzymes, such as urease, and exogenous ammonia act on phosphofructokinase activity shed new light on the regulatory mechanisms that govern glycolysis. Phosphofructokinase is the key enzyme known to exert a regulatory role on glycolytic flux and, therefore, ammonia as an effector of phosphofructokinase acts, in cascade, modulating the glycolytic pathway. Apart from S. thermophilus, due to the high conservation of glycolytic enzymes in all branches of the tree of life and being aware of the role of ammonia as an effector of phosphofructokinase, we propose to reevaluate the physiological role of the ammonia production pathways in all organisms whose energy metabolism is supported by glycolysis.