The adverse health effects of particulate matter less than 2.5 µm in diameter (PM2.5) have drawn increasing attention over the past several decades. To obtain reliable measurements, it is critical to efficiently separate PM2.5 in the airstream from the beginning till the end of sampling. However, commonly used separators for PM2.5 monitoring, such as the BGI Very Sharp Cut Cyclone (VSCC), are usually subject to aerosol-loading effects. This study investigates the loading effect on cyclone separation performance as a function of particle size, cyclone size, particle material, and air humidity. Based on the ratios of dimensions to the body diameter of the BGI VSCC, four cyclones with different body diameters (13–35.6 mm) were fabricated. An ultrasonic atomizer was employed to generate micrometer-sized potassium sodium tartrate (PST) particles and sodium chloride (NaCl) particles as solid challenge particles and di-ethyl-hexyl-sebacate (DEHS) particles as liquid ones. Aerosol particles were neutralized to the Boltzmann charge equilibrium. An aerodynamic particle sizer measured the aerosol distributions and number concentrations upstream and downstream of the cyclones. The experimental results show that solid particles such as PST with sizes close to the cyclone cut-point exhibit a significant loading effect. However, no significant difference is found due to the aerosol loading effect on four different-sized cyclones. The cyclone separation curve appears to shift toward smaller sizes due to aerosol loading. During the loading test, the aerosol penetration of 2.5-µm particles abruptly decreased during the first 20 minutes from 50% to a relatively stable level at 30%—an average decrease of 20%. Thus, the performance of cyclone PM2.5 samplers with progressive aerosol loading might result in an underestimation of PM2.5, particularly for continuous monitoring.