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The deposition of particles onto indoor surfaces can significantly affect people’s exposure to PM2.5 in buildings. Therefore, it is important to accurately determine indoor PM2.5 deposition for indoor air quality design, exposure assessment, and practical control. However, few, if any, studies on experimental measurement of PM2.5 deposition rate under different ventilation rates in buildings with natural ventilation have been reported. This study first experimentally investigated the influence of natural ventilation rate on PM2.5 deposition in two university classrooms in Nanjing, China. It was found that the deposition rate was positively associated with the natural ventilation rate for both classrooms. This study developed an empirical equation to correlate the PM2.5 deposition velocity with the natural ventilation rate. The developed empirical equation can be applied to test how the natural- ventilation-rate-dependent PM2.5 deposition velocity influenced the infiltration factor, I/O ratio, and air cleaner selections.