Serotonin (5-Hydroxytriptamine, 5-HT) has been implicated in the regulation of many physiological processes, such as cellular growth and differentiation and regulation of blood glucose. Plasma 5-HT concentrations increase under several physiological and pathological conditions, including fasting, diabetes and hypertension. It has been shown that 5-HT causes a rapid stimulation in glucose uptake by increasing the plasma membrane content of GLUT 1, 3 and 4 in rat and human skeletal muscle, which has been reported to express 5-HT_2A receptor. Additionally, it was suggested that 5-HT promotes its effects inducing a rapid and transient activation of protein phosphorylation. The purpose of this study is to analyze whether 5-HT modulates 6-phosphofructo-1-kinase (PFK) activity in mice skeletal muscle, recognized as the main enzyme controlling the glycolytic flux. Our experiments revealed that 5-HT up regulate PFK activity in a dose-dependent manner achieving its maximal effect at 25 pM 5-HT. Enzyme kinetics experiments revealed that 25 pM 5-HT increases both the V_max (161.7 ± 7.6 and 232.7 ± 38.0 mU/µg, for control and 25 pM 5-HT, respectively, P < 0.05, student’s t-test) and affinity for fructose-6-phosphate (K_m were 0,24 ± 0.07 and 0,09 ± 0,02 mM, for control and 25 pM 5-HT, respectively, P < 0.05, student’s t-test). In the attempt to investigate possible mechanisms of action by the hormone, we measured protein phosphorylation pattern in the skeletal muscle homogenate, where 25 pM 5-HT promoted a 75% increase of phosphate incorporated in total protein. Additionally, we found that upon stimulation with 25 pM 5-HT, PFK was 125 % more associated to cytoskeleton filaments, comparing to control. We have previously shown that phosphorylation of PFK increases its affinity to f-actin, which promotes the activation of the enzyme. Altogether, our results support evidences that 5-HT stimulates PFK activity in skeletal muscle through the phosphorylation of the enzyme, stabilizing its cytoskeleton-bound active configuration.