Abstract: Plant factories have been one of the most important research directions in modern agricultural engineering. Particularly, there is a year-round continuous production and precise environmental regulation. However, a stable and uniform internal environment is increasingly challenging, as the vertical and horizontal dimensions of the planting region. The thermal load can be caused by the continuous heat release from the artificial light sources. It is often required for the internal microclimate regulation in the large-scale plant factories. There are significant impacts of the disordered airflow, uneven temperature distribution, and localized heat accumulation on the growth of leafy vegetables. Conventional single circulation ventilation cannot fully meet the environmental control demands of the large-scale plant factories. Their airflow penetration can often fail to ensure the uniform temperature regulation across the planting region. This study aims to investigate a double circulation upward return air system with the bilateral horizontal air supply and vertical roof return air. A systematic analysis was made on the temperature distribution and flow field characteristics in the large-scale plant factory using the combination of actual measurement and CFD simulation. The actual measurement results demonstrated that the temperature difference in the 1 m/s horizontal air supply region reached up to 3.8 ℃, while the corresponding difference in the 10 m/s horizontal air supply region was 2.2 ℃. Horizontally, the temperature distribution exhibited higher temperatures in the center and lower temperatures on both sides, with the high temperature zone on the side of the 1 m/s horizontal air supply region. Vertically, the significant stratification was observed, with the heat accumulating in the upper layers. The maximum temperature difference across the entire planting area reached 3.8 °C, indicating a non-uniform temperature field. The CFD simulation results further revealed that the low-velocity airflow (1 m/s) from the left and the high-velocity airflow (10 m/s) from the right converged to form a vortex region, where the airflow velocity dropped below 0.3 m/s. This low-velocity zone restricted the heat dissipation, leading to the localized high-temperature zone. The main body of the airflow convergence zone was located in the low velocity air inlet regulation zone, occupying 16.9% of the area of the low velocity air inlet regulation zone. There was a difference in the air inlet velocity between the left and right sides, as well as the obstruction caused by facilities, such as the nutrient tank on the right side. Both sides were then adjusted to balance the large difference in the air inlet velocity between the left and right air inlet walls. Additional CFD simulations were carried out to promote the left-side inlet velocity to 1.5 and 2.0 m/s. Furthermore, the cold air penetration depth improved, and the vortex region gradually shifted rightward, as the velocity on the left side increased. The area of the vortex zone decreased to only 5.2% when the left-side inlet air velocity was 2 m/s. There was a 42% reduction in the vortex range, compared with the left-side inlet air velocity of 1 m/s. The thermal mass was reduced to uniformly distribute the temperature field across the entire space. This finding can also provide a strong reference for the ventilation systems in large-scale plant factories.