Burkholderia gladioli, primarily known as a plant pathogen, is involved in human infections, especially in patients with cystic fibrosis (CF). In the present study, the first respiratory isolates recovered from 14 French patients with CF and 4 French patients without CF, identified by 16S rRNA gene analysis, were tested for growth on B. cepacia selective media, for identification by commercial systems, and for their antimicrobial susceptibilities, and were compared by pulsed-field gel electrophoresis (PFGE). Patients' data were collected. All 18 isolates grew on oxidation-fermentation-polymyxin B-bacitracin-lactose medium and Pseudomonas cepacia agar, but only 13 grew on Burkholderia cepacia selective agar. API 20NE strips did not differentiate B. gladioli from B. cepacia, whereas Vitek 2 GN cards correctly identified 15 isolates. All isolates were susceptible to piperacillin, imipenem, aminoglycosides, and ciprofloxacin and were far less resistant to ticarcillin than B. cepacia complex organisms. Fifteen PFGE types were observed among the 18 isolates, but shared types were not identified among epidemiologically related patients. The microbiological follow-up of CF patients showed that colonization was persistent in 3 of 13 documented cases; B. gladioli was isolated from posttransplantation cultures of blood from 1 patient. Among the patients without CF, B. gladioli was associated with intubation (three cases) or bronchiectasis (one case). In summary, the inclusion of B. gladioli in the databases of commercial identification systems should improve the diagnostic capabilities of those systems. In CF patients, this organism is more frequently involved in transient infections than in chronic infections, but it may be responsible for complications posttransplantation; patient-to-patient transmission has not been demonstrated to date. Lastly, B. gladioli appears to be naturally susceptible to aminoglycosides and ciprofloxacin, although resistant isolates may emerge in the course of chronic infections.