Limited available land for livestock farming and increasing land-use costs have forced some large animal feeding operations to build animal barns from conventional one-floor to multi-floor in China recently because multi-floor animal building (MFAB) has a great advantage in saving land utilization. The ventilation system is an important part of animal building determining the indoor environmental condition. The difference in ventilation rate between MFAB and conventional barn might exist, because of the difference in building structure, especially due to the confined space outside of exhaust fans in the MFAB. However, a limited number of studies have accumulated information on the ventilation performance of this novel multi-floor animal barn. This work aims to acquire such information relating to the ventilation performance of a novel MFAB with a different number of floors (6, 7, 8, 9, and10 floors building) and different width of the yard using the Computational Fluid Dynamics (CFD) technique. A field measurement was carried out in a six-floor pig barn, and the measured air velocities were used for the CFD model validation. The validated CFD model was then extended to explore the difference of airflow patterns and ventilation rates on different floors under a different number of floors and the width of the yard. The simulation results showed that the difference in airflow pattern among each floor was limited, but the ventilation rate of each floor varied from floor to floor under the studied type of MFAB. The ventilation rate on the first floor was the lowest and increased with the floor increasing. Hence, more focus on the ventilation rate of the first floor should be paid during the design procedure. The increase of the floor number negatively affected the performance of the ventilation system because of the pressure profile in the yard of the building. Increasing the width of the yard could mitigate the negative effect of the floor number on the ventilation rates. Therefore, to perform the desired ventilation rate for animals in a multi-floor animal building required a more detailed design of the ventilation system of each floor by considering either the space available after exhaust openings or additional fans.