<p><b>Abstract</b><br>Thermogenic adipocytes have been extensively investigated due to their energy dissipating property and therapeutic potential for obesity and diabetes. Besides serving as fuel sources, accumulating evidence suggests that intermediate metabolites play critical roles in multiple biological processes. However, their role in adipocyte differentiation and thermogenesis remains unexplored. Here, we report that human and mouse obesity is associated with marked downregulation of glutamine synthetase (Glul) expression and activity in thermogenic adipose tissues. Glul is robustly upregulated during brown adipocyte (BAC) differentiation and in brown adipose tissue (BAT) upon cold exposure and Cl316,243 stimulation. Further genetic, pharmacologic, or metabolic manipulations of Glul and glutamine levels reveal that glutamine cell-autonomously stimulates BAC differentiation and function, BAT tissue remodeling, and improves systemic energy homeostasis in mice. Mechanistically, glutamine promotes transcriptional induction of adipogenic and thermogenic gene programs through histone modification-mediated chromatin remodeling. Among all the glutamine-regulated writer and eraser genes responsible for histone methylation and acetylation, only Prdm9, a histone lysine methyltransferase, is robustly induced during BAC differentiation. Importantly, Prdm9 inactivation by shRNA knockdown or a selective inhibitor attenuates glutamine-triggered adipogenic and thermogenic induction. Further, Prdm9 gene transcription is regulated by glutamine through the recruitment of C/EBPb to its enhancer region. This work reveals glutamine as a novel activator of thermogenic adipocyte differentiation and uncovers an unexpected role of C/EBPb-Prdm9-mediated H3K4me3 and transcriptional reprogramming in adipocyte differentiation and thermogenesis.</p><p><br></p><p><b>Article Highlight:</b></p><p>• Glutamine is a pivotal player in multiple biological processes and disease progression. However, its role in thermogenic adipocyte differentiation and function remains unclear.</p><p>• Through genetic, pharmacologic, or metabolic manipulations of Glul and glutamine levels, we investigated the role and mechanism of Glul/glutamine pathway in adipocyte thermogenesis and systemic homeostasis.</p><p>• We revealed that Glul-mediated glutamine production is critical for BAC differentiation, and uncovered an unexpected role of C/EBPb-Prdm9-mediated histone methylation and transcriptional reprogramming in this process.</p><p>• This work highlights the therapeutic and translational potential of targeting the Glul/glutamine/Prdm9 pathway to treat obesity and associated metabolic diseases.</p>