Background <p><i>Selenomonas sputigena</i> is an anaerobic mucosa-associated bacterium with dual roles in human health—acting as a pathobiont in periodontal disease and exhibiting protective effects in allergic airway inflammation. Despite its clinical significance, its metabolic functions and underlying mechanisms remain poorly defined.</p> Objective <p>This study aimed to systematically characterize <i>S. sputigena’s</i> metabolic capacity and develop genetic tools for functional studies.</p> Methods <p>We reconstructed central carbon metabolic networks through in silico analysis. Growth kinetics, substrate utilization, and fermentation profiles were evaluated experimentally across five carbon sources: glucose, gluconate, glycerol, glutamate, and succinate. Expression of key metabolic genes was quantified by qRT-PCR. Native promoter constructs were developed and tested for GFP reporter expression.</p> Results <p><i>Selenomonas sputigena</i> displayed glucose-preferential growth with rapid consumption (0.70 ± 0.05&#xa0;mM&#xa0;h⁻¹) and substantial acetate production (17.76 ± 2.05&#xa0;mM). Gluconate and glycerol supported moderate growth, while glutamate and succinate were poorly utilized. Gene expression analysis revealed strong substrate-dependent regulation of glycolytic genes, with <i>gap</i> expression correlating with growth performance, while TCA cycle genes maintained constitutive basal expression. Four native promoters successfully drove reporter expression, with P_<i>gap</i> demonstrating superior performance as a growth-responsive reporter.</p> Conclusions <p>This study establishes the first comprehensive metabolic and genetic framework for <i>S. sputigena</i>, revealing glucose-dependent fermentation with high acetate production that may contribute to host interactions. The validated promoter system enables future investigations of host–microbe interactions and therapeutic applications in mucosal environments.</p>