Natural ventilation is energy-saving and preferred in livestock buildings under suitable conditions, especially for cattle and sheep. The natural ventilated livestock buildings usually have large-openings which result in the non-uniform distribution of wind speed and pressure at the openings, which makes it difficult to determine the air inlet and outlet and challenging to estimate airflow rate in a natural ventilated livestock buildings. Thermal buoyancy and wind pressure are the driving forces of natural ventilation whose airflow rate can be estimated directly by differential pressure method, wind speed method and CFD method, or indirectly by heat balance, moisture balance, CO2 balance and tracer gas method. This review paper investigates the published works on the theory of natural ventilation in livestock buildings and the methods of airflow rate estimation to obtain a better understanding on the state of art for the mechanisms and estimation methods of airflow rate of natural ventilation in livestock buildings. Furthermore, this paper summarizes the key factors affecting natural ventilation and the principles of improving accuracy in the application of estimation methods to guide ventilation measurements better, promote natural ventilation or mixed ventilation in livestock barns, and provide a basis for optimizing environmental control strategies for the environment as a whole. Finally, different estimation methods of airflow rate are compared and the research gaps need to be further improved are concluded in this paper. Up to now, it is short of an accurate and disputable method to measure airflow rate in natural ventilation. The differences among different estimation methods are generally between 10% and 300%, showing a very big variance. The estimation accuracy of different methods is affected by the factors such as the discharge coefficient, the pressure coefficient, the reliability of estimated heat and moisture production from animal, and the measuring position of sensors. Compared with other methods, the CO2 balance method is widely used in the field measurements, and the result is relatively stable. However, it is highly needed to standardize the layout of sensors and the usage of the measured gas concentrations from different sensors to appropriately calculate concentration difference between indoor and outdoor, improving the accuracy of assessing CO2 production from livestock buildings. The moisture balance method has a potential application in field measurements and practical situations because of its low cost of sensors and relatively good performance in estimation airflow rate. Nevertheless, it is necessary to further establish the estimation models on moisture production from house level and dynamic latent heat production from animals to improve the accuracy of moisture balance method. In addition, it is helpful to assess the reliability of the measured airflow rates and decrease the uncertainty in airflow rate estimation using multiple methods at the same time. Furthermore, the existing methods are more suitable for design assistance rather than regulating the airflow rate in real situation. It is necessary to improve the existing method or establish a new method to guide the regulation of natural ventilation directly and effectively in livestock buildings. © 2020, Editorial Department of the Transactions of the Chinese Society of Agricultural Engineering. All right reserved.