Simple SummaryAlthoughL. delbrueckiistrains have been isolated from different bee products and beehive niches to our knowledge, this is the first report of the isolation of theL. delbrueckiistrain from the gut of a honeybee. Furthermore, the genomic analysis of the strain revealed two major aspects of its genomic constitution: (1) it possesses many genetic determinants attributing probiotic properties for the honeybees, and (2) it shows a reduction in size which is a typical adaptation of bacteria undergoing transformation to an endosymbiont. These two observations motivated us to hypothesize that in the case ofL. delbrueckiisubsp.lactisA4, we witness the first case of anL. delbrueckiistrain evolution to a honeybee endosymbiont.AbstractALactobacillus delbrueckiissp.lactisstrain named A4, isolated from the gut of an Armenian honeybee, was subjected to a probiogenomic characterization because of its unusual origin. A whole-genome sequencing was performed, and the bioinformatic analysis of its genome revealed a reduction in the genome size and the number of the genes—a process typical for the adaptation to endosymbiotic conditions. Further analysis of the genome revealed thatLactobacillus delbrueckiissp.lactisstrain named A4 could play the role of a probiotic endosymbiont because of the presence of intact genetic sequences determining antioxidant properties, exopolysaccharides synthesis, adhesion properties, and biofilm formation, as well as an antagonistic activity against some pathogens which is not due to pH or bacteriocins production. Additionally, the genomic analysis revealed significant potential for stress tolerance, such as extreme pH, osmotic stress, and high temperature. To our knowledge, this is the first report of a potentially endosymbioticLactobacillus delbrueckiissp.lactisstrain adapted to and playing beneficial roles for its host.