Abstract Type 2 Diabetes Mellitus (T2DM) is a chronic metabolic disorder characterized by insulin resistance and β-cell dysfunction. Glucagon-like peptide-1 (GLP-1) has emerged as a pivotal therapeutic target due to its roles in promoting insulin secretion, regulating appetite, and enhancing lipid metabolism. However, GLP-1-based therapies face significant challenges, including high production costs, complex delivery methods, and rapid in vivo degradation. To address these limitations, we developed a novel gut delivery strategy using Bacillus subtilis PY79 as a host for the expression of pentameric GLP-1. The genetically-modified strain, B. subtilis JH389, is demonstrated to maintain the same probiotic and antibiotic-sensitivity features belonged to the original strain PY79, and was deposited at the BCCM/LMG collection as B. subtilis LMG P-34037. The pentameric GLP-1 was designed to be digested by intestinal trypsin and to release active GLP-1 monomers capable of (i) resisting inactivativation by DPP-4 and trypsin, (ii) crossing the intestinal mucosa and (iii) entering the bloodstream. SDS-PAGE and Western blot confirmed the expression of pentameric GLP-1 in B. subtilis JH389, and fecal sample analysis demonstrated the strain gut engraftment. The administration of B. subtilis JH389 increased serum GLP-1 concentration, while also reducing blood glucose levels in a mouse model. Our in vitro and in vivo studies highlights the therapeutic potential of the JH389 strain and support the feasibility of using genetically engineered bacteria as a cost-effective and efficient delivery platform for GLP-1, paving the way for future applications in diabetes and metabolic disorder treatments.