In the sputum culture of a patient with tracheotomy, we found a kind of Gram-negative bacteria with membrane growth, which was not identified by Vitek2 Compact. Then, the strain was identified by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and 16S ribosomal RNA (16S rRNA) sequencing, and the results showed that the strain was Kerstersia gyiorum. Kerstersia gyiorum is a kind of Gram-negative non-fermenting bacterium. A few of foreign cases reported that this bacterium might be a conditional pathogen, which could be isolated from sputum and alveolar lavage fluid of tracheotomy patients, and from urine of percutaneous fistula patients. This bacterium could also cause bacteremia and participate in the occurrence and development of chronic otitis media. Chinese literature on the isolation of Kerstersia gyiorum is rare. The strain of the bacterium was isolated, and the basic characteristics of the bacterium were reported as follows to enable clinical microbial laboratory personnel to strengthen the understanding and attention on it.