Air distribution is an important factor affecting the flow field of a laboratory animal room in a barrier environment. We studied a mouse breeding room in a barrier environment as an example, using Airpak software to simulate the effect of the position, number and size of exhaust outlets, as well as the cage position, on the indoor flow field. The simulation result showed that the number and location of exhaust outlets mainly affected the local air distribution, and had little effect on the average temperature, air speed, air age and mean square deviation of the entire mouse feeding room. The average age of air particles in cages arranged along the width direction of the room was 25 s shorter than that in cages arranged along the length direction of the room. Additionally, there was a linear function relationship between the fresh air cooling load index during summer air conditioning and the number of air changes, with the fresh air cooling load accounting for 92. 25% of the total cooling load. Compared with the open cage system, the fresh air cooling load index of the independent ventilation cage system was reduced by 62. 6%.