Loess is an aeolian, nonstratified deposit. Loess collapsibility is considered to be closely related to its microstructure. The three-dimensional (3D) microstructure of loess specimens was established based on serial X-ray computed tomography (CT) images with a voxel size of 1 μm3. The loess microstructure and parameters, including the particle size, sphericity, and orientation and pore size distribution were characterized qualitatively and quantitatively in 3D space. In addition, the microstructural evolution during collapse based on the 3D microstructure was also analyzed to interpret the mechanism of loess collapse combined with X-ray diffraction and scanning electron microscopy results. The results highlight that loess collapsibility originates from both microstructural and compositional characteristics. The studied loess has an open structure with widely distributed spaced pores and inter-particle or inter-aggregate pores. Brick-shaped and subangular particles with tilted orientations and point-to-point contacts maintain a vulnerable, metastable structure. Carbonate and clay aggregations between particles endow the loess with a high strength in the natural state. Under loading and wetting, the swelling and dispersion of clay aggregation, particularly the degraded illite and interstratified illite - montmorillonite, and the loss of high suction initiate structural failure. During collapse, particles primarily move down in a vertical or tilted direction with little rotation. Pores are most sensitive to loading and wetting; during collapse, the distributions of the pore number and volume percentage become more unimodal. The pore connectivity becomes weaker. Pores larger than 23 μm decrease in pore number under wetting, and pores larger than 26 μm decrease in volume percentage.