Purpose <p>This study aimed to develop a novel semaglutide-loaded dry powder inhaler (DPI) with improved bioavailability compared with oral administration while avoiding discomfort associated with injection.</p> Methods <p>To determine the optimal process for preparing micronized semaglutide powder, pure semaglutide, spray-dried semaglutide and freeze-dried semaglutide were compared. The effects of lactose, sucrose, and mannitol on the freeze-drying process, as well as the effect of lactose carrier amount on aerodynamic performance of DPI were evaluated. The morphology, impurity levels, aggregate formation, and aerodynamic particle size distribution of semaglutide-loaded DPI were assessed.</p> Results <p>Freeze-drying produced micronized particles with elongated, needle-like shape, without significantly affecting the impurity and aggregation profiles of semaglutide. The optimized semaglutide-loaded DPI, composed of semaglutide, mannitol, coarse lactose, fine lactose, and magnesium stearate at a weight ratio of 0.71:2.0:2.203:0.55:0.037, achieved a fine particle mass (FPM) of 36.9% of the total semaglutide, representing the fraction of particles with an aerodynamic diameter smaller than 5&#xa0;μm suitable for deep lung deposition. Furthermore, intratracheal administration of semaglutide-loaded DPI in rats resulted in a relative bioavailability of 15.7%, which was higher than that of subcutaneous injection, and demonstrated a significant improvement over commercial oral tablet.</p> Conclusion <p>These findings highlight the potential of DPI as an effective alternative to injectable and oral semaglutide and offer a promising approach for improved semaglutide delivery.</p>