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Three-dimensional printing (3DP) offers valuable insight into the characterization of natural rocks and the verification of theoretical models due to its high reproducibility and accurate replication of complex defects such as cracks and pores. In this study, 3DP gypsum samples with different printing directions were subjected to a series of uniaxial compression tests with in situ micro-computed tomography (micro-CT) scanning to quantitatively investigate their mechanical anisotropic properties and damage evolution characteristics. Based on the two-dimensional (2D) CT images obtained at different scanning steps, a novel void ratio variable was derived using the mean value and variance of CT intensity. Additionally, a constitutive model was formulated incorporating the proposed damage variable, utilizing the void ratio variable. The crack evolution and crack morphology of 3DP gypsum samples were obtained and analyzed using the 3D models reconstructed from the CT images. The results indicate that 3DP gypsum samples exhibit mechanical anisotropic characteristics similar to those found in naturally sedimentary rocks. The mechanical anisotropy is attributed to the bedding planes formed between adjacent layers and pillar-like structures along the printing direction formed by CaSO4·2H2O crystals of needle-like morphology. The mean gray intensity of the voids has a positive linear relationship with the threshold value, while the CT variance and void ratio have concave and convex relationships, respectively. The constitutive model can effectively match the stress–strain curves obtained from uniaxial compression experiments. This study provides comprehensive explanations of the failure modes and anisotropic mechanisms of 3DP gypsum samples, which is important for characterizing and understanding the failure mechanism and microstructural evolution of 3DP rocks when modeling natural rock behavior.