Ventilation system is crucial for poultry houses to control the indoor climate and air quality. The tunnel ventilation system is widely applied for large-scale poultry buildings in China but only limited scientific researches regarding the flow pattern, temperature distribution and design criteria are available in the literature. Thanks to the fast development of computer technology, Computational Fluid Dynamics (CFD) techniques were used in present study to investigate the indoor air movement, air temperature and relatively humidity. A three-dimensional CFD model was built according to the real dimensions of a laying hen house and the model was validated by comparing the simulation results with the field measurements at 30 positions. Meanwhile, statistical analysis was performed to determine the differences between different boundary conditions regarding the agreement between measured and CFD simulated results. Optimization of air inlet configurations was performed by using the validated CFD model and it was found that the uniformity of indoor air movement could prevent excessive local convective heat losses and reduce the temperature at the end of the house. Furthermore, the air inlets placed at the middle of the side wall could significantly reduce the high temperature expected at the end of the building without using extra energy, which is especially important for large-scale poultry farms with long buildings. The performance of side-wall windows was also examined and preliminary guidance was provided to effectively regulate the indoor climate by using these windows with the help of environmental monitoring systems. The present study contributes to the understanding and design of the tunnel ventilation system used in poultry houses.