Object Mycoplasma pneumoniae (MP) is one of the key pathogens responsible for respiratory diseases in rats. In this study, the pathogen was isolated and identified from a rat farm experiencing respiratory disease outbreaks, followed by pathogenicity experiments in rats to identify the currently circulating strain. Methods PCR detection was performed on lung tissues from rats suspected to be infected with MP, yielding an amplified single band of approximately 280 bp. The samples were inoculated into mycoplasma broth medium and further purified on mycoplasma agar plates. The isolate was identified based on colony morphology, biochemical characteristics, amplification of the 16S rRNA gene, and homology analysis. For the pathogenicity test, 5-week-old SD rats were intranasally inoculated with a bacterial suspension at a concentration of 1×10? CCU/ml, and their clinical symptoms and pathological changes were observed. Results The PCR results preliminarily confirmed that the suspected cases were infected with Mycoplasma pulmonis. The growth characteristics in mycoplasma broth medium matched those of the target organism, and after purification, the colonies appeared round and stained light purple following Giemsa staining. Biochemical tests revealed acid production from glucose fermentation but no hydrolysis of arginine or urea. The amplification and sequencing of the 16S rRNA gene showed a high homology (99.05%) with the rat-derived MP strain (AL445565.1), confirming the successful isolation of Mycoplasma pulmonis. After challenging SD rats with the isolate, respiratory symptoms were observed, including pulmonary hemorrhage, consolidation, massive infiltration of inflammatory cells, and positive MP PCR results in lung tissues. Conclusion This study successfully isolated a strain of Mycoplasma pulmonis from rats, which induced typical pneumonia symptoms in SD rats. These findings provide a solid foundation for further research into the prevention and control of mycoplasma pneumonia in rats.