Abstract The most common form of epilepsy among adults is mesial temporal lobe epilepsy (mTLE), with seizures often originating in the hippocampus due to abnormal electrical activity. The gold standard for the histopathological analysis of mTLE is histology, which is a two-dimensional technique. To fill this gap, we propose complementary three-dimensional (3D) X-ray histology. Herein, we used synchrotron radiation-based phase-contrast microtomography with 1.6 μm-wide voxels for the post mortem visualization of tissue microstructure in an intrahippocampal-kainate mouse model for mTLE. We demonstrated that the 3D X-ray histology of unstained, unsectioned, paraffin-embedded brain hemispheres can identify hippocampal sclerosis through the loss of pyramidal neurons in the first and third regions of the Cornu ammonis as well as granule cell dispersion within the dentate gyrus. Morphology and density changes during epileptogenesis were quantified by segmentations from a deep convolutional neural network. Compared to control mice, the total dentate gyrus volume doubled and the granular layer volume quadrupled 21 days after injecting kainate. Subsequent sectioning of the same mouse brains allowed for benchmarking 3D X-ray histology against well-established histochemical and immunofluorescence stainings. Thus, 3D X-ray histology is a complementary neuroimaging tool to unlock the third dimension for the cellular-resolution histopathological analysis of mTLE.