Rationale and Objectives: Capillarization of sinusoids and change of trabecular thickness are the main histologic features in hepatocellular carcinoma (HCC). Of particular interest are the three-dimensional (3D) visualization and quantitative evaluation of such alterations in the HCC progression. X-ray phase-contrast computed tomography (PCCT) is an emerging imaging method that provides excellent image contrast for soft tissues. This study aimed to explore the potential of in-line PCCT in microstructure imaging of capillarized sinusoids and trabecular structure in human HCC tissues and to quantitatively evaluate the alterations of those fine structures during the development of HCC. Materials and Methods: This project was designed as an ex vivo experimental study. The study was approved by the institutional review board, and informed consent was obtained from the patients. Eight human resected HCC tissue samples were imaged using in-line PCCT After histologic processing, PCCT images and histopathologic data were matched. Fine structures in HCC tissues were revealed. Quantitative analyses of capillarized sinusoids (ie, percentage of sinusoidal area [PSA], sinusoidal volume) and trabecular structure (ie, trabecular thickness, surface-area-to-volume ratio [SA/V]) in low-grade (well or moderately differentiated) and high-grade (poorly differentiated) HCC groups were performed. Results: Using PCCT, the alterations of capillarized sinusoids and trabecular structure were clearly observed in 3D geometry, which was confirmed by the corresponding histologic sections. The 3D qualitative analyses of sinusoids in the high-grade HCC group were significantly different (P < 0.05) in PSA (7.8 +/- 2.5%) and sinusoidal volume (2.9 +/- 0.6 x 10(7) mu m(3)) from those in the low-grade HCC group (PSA, 12.9 +/- 2.2%; sinusoidal volume, 2.4 +/- 0.3 x 10(7) mu m(3)). Moreover, the 3D quantitative evaluation of the trabecular structure in the high-grade HCC group showed a significant change (P < 0.05) in the trabecular thickness (87.8 +/- 15.6 mu m) and SAN (2.2 +/- 1.3 x 10(3) mu m(-1)) compared to the low-grade HCC group (trabecular thickness, 75.9 +/- 7.1 mu m; SA/V, 7.5 +/- 1.3 x 10(3) mu m(-1)). Conclusions: This study provides insights into the 3D alterations of microstructures such as capillarized sinusoids and the trabecular structure at a micrometer level, which might allow for an improved understanding of the development of HCC.