Objective The present study was designed to establish a model of mycoplasma pneumonia of swine ( MPS) by artificial challenge. Methods Mycoplasma hyopneumoniae Is strain was isolated and identified by biochemical and serological assays. MPS negative pigs were inoculated with 2ml of Mycoplasma hyopneumoruae is strain culture by direct lung inoculation. The pigs were observed daily for 15 to 20 days post-inoculation for clinical symptoms and pathological changes. Infected tissues with live Mycoplasma hyopneumoniae virulent strain were lyophilized and used as the master seed for animal challenge. Artificial challenge was performed by delivering 5ml of lyophilized pathological tissues, intratracheally,into 15 Meishan pigs. The lyophilized pathological tissues used for challenge were diluted by 10^-2,10^-3, 10^-4 fold in KM₂ culture medium, and pigs in the control group were inoculated with 5 ml KM₂ culture medium. Clinical symptoms, pathological changes and lung X-ray results were observed twenty-five days post challenge. Results Typical clinical symptoms and pathological changes associated with MPS were observed in all pigs intratracheally inoculated with lyophilized pathological tissues that were diluted in KM₂ culture medium. Therefore, 5 ml of the 10^-4 dilution of lyophilized pathological tissues with Mycoplasma hyopneumoniae is strain was the minimum dose required to induce MPS in an artificial challenge model. ConeJusion The artificial challenge model of MPS coWd be established by intratracheally inoculation of 5m1 of a 1o-2 dilution of lyophilized pathological tissues infected with the Myooplasma hyopneumoniae is strain.