Background and aims: Glutamine synthetase (GS), a key enzyme in the glutamine synthesis, is thus crucial in glutamine homeostasis. GS is known to be up-regulated by fasting and inhibited by glutamine supplementation. The aim of this study was to determine whether the presence of glucose in glutamine supplementation with refeeding differently affects up-regulation of muscle GS by fasting in vivo in adult female rats than glutamine alone. Methods: Muscle GS activities were assessed in 5-day-fasted female Wistar adult rats refed and supplemented with glutamine or glycine in the presence or not of glucose. Results: After 5-day-fasting, the up-regulated GS activity was decreased whatever the type of amino acid supplementation (glutamine or glycine), whereas it was more decreased by supplementation with a mixture glutamine/glucose. In glycine/glucose supplemented rats, no effect of glucose supplementation was observed on GS activity. Conclusion: These results demonstrated that intramuscular glutamine was spared when glucose was added to glutamine supplementation in adult rats. Consequently, the role of glucose consisted in slowing down the glutamine synthesis. By contrast, glucose has no role when it was associated with glycine whose degradation does not produce energy.