Abstract: The aim of this study was to establish a high-density fermentation process of Mycoplasma hyopneumoniae HN0613 strain （Mhp HN0613）. The passage number with best growth activity and the optimum recovery time of Mhp HN0613 were determined using CCU as assessing index; a four-factor and three-level orthogonal test was conducted to screen the optimum basal culture medium formula; the aeration rate, agitation speed and inoculation percentage were used as single evaluating factor to optimize the parameters of fermentation process with volume of 15 L, respectively; based on the developed 15 L high-density fermentation process, a 700 L scaleup fermentation process was subsequently developed; the immune efficacy of the inactivated vaccine of Mhp HN0613 prepared with the developed high-density fermentation process on rabbit and piglet was evaluated. The results showed that the best growth activity was observed in F11 of Mhp HN0613 with CCU of 1×10^9 CCU/mL; the pH value of the medium of Mhp HN0613 reduced to around 7.0 at 72 h post recovery, and the CCU of Mhp HN0613 reached up to the highest level of 1×10^9 CCU/mL, accordingly, 72 h was considered as the optimum recovery time; the optimized basal culture medium formula was as follows： 2% of initial addition of glucose, 8% of inoculation percentage of Mhp HN0613, 7.6 of pH value of the initial culture medium, and 15% of addition of porcine serum; the optimized the parameters of 15 / fermentation process of Mhp HN0613 was as follows： 100 L/h of aeration rate, 300 r/min of agitation speed, and 8% inoculation percentage; a process technology route of balance k,a linkage pH value back-regulation was initially developed in this study; the agitating impeller in the fermentation tank was altered by impulse impeller, which reduced the shear stress of impeller against the bacteria, and the CCU of Mhp HN0613 was increased ten times and the culture period was shorted by 2-3 days; in the 700 L fermentation scaleup process, the culture titer of Mhp HN0613 was no less than 5x 109 CCU/mL and even reached up to the highest level of 1×10^10 CCU/mL; the serum antibody titer of the immunized rabbits with Mhp HN0613 was no less than 1：40 and the highest level was observed as 1：160; the pneumonia decreasing rate of the immunized piglets were all above 80%, and all of the immunized piglets were clinically healthy. This study provides a theoretical basis for the large-scale Mhp vaccine production.