[Objective] This study aimed to investigate the effect of hypertonic solution on the morphology,size,incubation time and cell viability of Mycoplasma hyopneumoniae(Mhp).[Method] Mhp was stimulated by using NaCl hypertonic solution with a final concentration of 1.8% for 1,2,4,6 and 8 min,respectively.After staining and microscopic examination,the particle size and CCU(color change unit) were determined,and PCR identification was conducted.[Result] After treated with hypertonic solution for different time,Mhp was shrunken and turned round,the particle size was reduced,and the cell number rapidly decreased or even disappeared after 2 min.CCU was reduced by a gradient,and the observation time was extended for 1 d.The target band of Mhp could be amplified from samples after treated with hypertonic solution for different time.After treated with 1.8% NaCl solution for 1-6 min,Mhp changed in the morphology and size but still had viability.[Conclusion] This study provided reference data for exploring the entrance of Mhp into blood circulation after intramuscular injection and a new immunization route of swine Mycoplasma pneumonia vaccine.