Along with the increasing attempts to explore the wound healing effective substances of Periplaneta americana (L.) (PA), a medicinal insect in traditional Chinese medicine, researchers' attention turned to the endogenetic macromolecules, such as polysaccharides and peptides. Herein, we innovatively isolated two glycoproteins from PA, named PAGP-1 and PAGP-2, which were obtained by Cellulose DE-52 chromatography and purified by Sephadex G-100 gel in succession. The structural characterization of the two PAGPs were performed, including molecular weight, amino acid and monosaccharide composition, morphology analysis, FT-IR and 1H NMR analysis, CD spectroscopy, and glycosides linkage. As a result, two PAGPs belonged to O-glycopeptide bonds linked glycoproteins. The content of carbohydrate and protein of PAGP-1 was approximately 25.23% and 65.92% respectively, which of PAGP-2 was approximately 25.71% and 71.23%. Based on the remarkable anti-inflammatory effects of PAGPs on LPS-induced RAW264.7 cells, the topical administration of PAGP-1 and PAGP-2 could significantly accelerate full-thickness wound healing in diabetic mice, involving to alleviate the inflammation, increase the ratio of type I and type III collagen fibers, and promote the polarization of macrophages M1 to M2. In short, this study provides clear evidence that the glycoproteins would be the potential wound healing bioactive substances in PA.