Objective Mycoplasma pneumoniae (MP) was isolated from a rat farm with respiratory diseases and the pathogen was identified. A rat pathogenicity experiment was conducted to obtain the current prevalent pathogen. Methods Lung tissues were isolated from rats with suspected MP infection and analyzed by PCR, 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. Pathogenicity tests were conducted in 5-week-old SD rats by intranasal inoculation with a bacterial suspension at a concentration of 1 × 10⁷CCU/ mL, and the subsequent clinical symptoms and pathological changes were observed. Results PCR confirmed that the suspected cases were infected with MP. The growth characteristics in mycoplasma broth medium matched those of the target organism, and the purified colonies appeared round and stained light purple with Giemsa stain. Biochemical tests revealed acid production from glucose fermentation but no hydrolysis of arginine or urea. Amplification and sequencing of the 16S rRNA gene showed high homology (99. 05%) with the rat-derived MP strain (AL445565. 1), confirming successful isolation of MP. Rats challenged with the isolate developed respiratory symptoms, including pulmonary hemorrhage, consolidation, massive infiltration of inflammatory cells, and positive MP PCR result in lung tissues. Conclusions This study successfully isolated a strain of MP 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 MP in rats.