Enterobacter sakazakii is an opportunistic food-borne pathogen causing meningitis, enterocolitis, and sepsis, primarily in immunocompromised infants. It has been suggested that stable flies, Stomoxys calcitrans L., are a vector/reservoir of this pathogen. In this study, we assessed a) vector competence of adult stable flies (SF) for E. sakazakii, b) effect of E. sakazakii on SF development, and c) survival of E. sakazakii during SF development and colonization of the digestive tract of newly-emerged flies. Our data show that in the colony, adult SF can maintain E. sakazakii for at least 20 days regardless of the food source (blood or sugar) and contaminate the food source. The concentration of the pathogen per individual SF ranged from 1.8 × 10⁵ to 6.4 × 10⁶ CFU. E. sakazakii supported development of immature SF in sterilized cattle manure and sterilized artificial medium (78.3% and 76.7% SF survival to adult stage, respectively). In addition, E. sakazakii survived during SF development and colonized the gut of emerging adult SF but only when SF larvae were maintained on sterilized cattle manure inoculated with E. sakazakii (12.8% prevalence in adult SF) and on the sterile artificial medium with E. sakazakii (21.7% prevalence in adult SF). E. sakazakii was not recovered from flies or the substrate when larvae were reared on cattle manure with a non-sterilized complex microbial community plus the E. sakazakii inoculum. This study shows that SF adults have a potential to carry E. sakazakii for an extended period of time. E. sakazakii supports SF development and can survive during SF pupation and then colonize the gut of newly-emerged flies.