Dietary restriction (DR) is one of the most robust interventions shown to extend health-span and remains on the forefront of anti-aging intervention studies, though conflicting results have been shown on its effect on lifespan both in rodents and primates. The severe inhibitory effects on the lymphoid lineage by DR remains one of its major negative downsides which reduces its overall beneficial effects on organismal health. Yet, the underlying mechanism of how DR suppresses the lymphoid system remains to be explored. Here, we show that antibiotic ablation of gut microbiota significantly rescued the inhibition of lymphopoiesis by DR. Interestingly, glycolysis in lymphocytes was significantly down-regulated in DR mice and pharmacological inhibition of glycolysis reverted this rescue effect of lymphopoiesis in DR mice with ablated gut microbiota. Furthermore, DR remarkably reconstructed gut microbiota with a significant increase in butyrate-producing bacterial taxa and in expression of But, a key gene involved in butyrate synthesis. Moreover, supplemental butyrate feeding in AL mice suppressed glycolysis in lymphoid cells and mimicked the inhibition of lymphopoiesis in AL mice. Together, our study reveals that gut microbiota mediates the inhibition on lymphopoiesis via down-regulation of glycolysis under DR conditions, which is associated with increased butyrate-synthesis. Our study uncovered a candidate that could potentially be targeted for ameliorating the negative effects of DR on lymphopoiesis, and therefore may have important implications for the wider application of DR and promoting healthy aging.