Due to the two major physiological barriers restricted by mucus penetration and epithelia transport, oral insulin therapy using nano-delivery system remains challenging. Heyndrickxia coagulans spores can survive the harsh conditions of gastrointestinal tract (GIT), and penetrate in the mucus through germination to probiotics with their amphipathic proteinaceous coat shedding in the gut epithelium, which makes it possible to be functionalized with hydrophilic peptide/protein and form nanoparticles (NPs) in vivo. Inspired by the natural physiological properties of spores, novel deoxycholic acid-modified Heyndrickxia coagulans spores loaded with insulin (DA-Spore/Ins) as the generators of autonomous bio-based nanoparticles were designed to solve these absorption barriers to enhance oral insulin delivery. The DA-Spore/Ins delivery system achieved preferable drug protection and rapid mucus penetration through its germination in the intestinal microenvironment. Meanwhile, DA-Spore/Ins NPs could be spontaneously formed by the self-assembly of the disintegrated DA-covalently amphipathic protein coat and the hydrophilic protein/peptides drug. This can efficiently transport through the epithelial cells through the bile acid pathway. In vivo studies indicated that DA-Spore/Ins delivery system achieved an oral relative bioavailability of 15.1 % and superior hypoglycemic effect in type I diabetic rats characterized by good biocompatibility. These studies suggested that the intrinsic biological characteristics of Heyndrickxia coagulans spore-based nanogenerators rendered their promising application in oral insulin or other protein drug therapy.