Diffusion microstructural imaging techniques have attracted great interest in the last decade due to their ability to quantify axon diameter and volume fraction in healthy and diseased human white matter. The estimates of compartment size and volume fraction continue to be debated, in part due to the lack of a gold standard for validation and quality control. In this work, we validate diffusion MRI estimates of compartment size and volume fraction using a novel textile axon (“taxon”) phantom constructed from hollow polypropylene yarns with distinct intra- and extra-taxonal compartments to mimic white matter in the brain. We acquired a comprehensive set of diffusion MRI measurements in the phantom using multiple gradient directions, diffusion times and gradient strengths on a human MRI scanner equipped with maximum gradient strength (Gmax) of 300 mT/m. We obtained estimates of compartment size and restricted volume fraction through a straightforward extension of the AxCaliber/ActiveAx frameworks that enables estimation of mean compartment size in fiber bundles of arbitrary orientation. The voxel-wise taxon diameter estimates of 12.2 ± 0.9 μm were close to the manufactured inner diameter of 11.8 ± 1.2 μm with Gmax = 300 mT/m. The estimated restricted volume fraction demonstrated an expected decrease along the length of the fiber bundles in accordance with the known construction of the phantom. When Gmax was restricted to 80 mT/m, the taxon diameter was overestimated, and the estimates for taxon diameter and packing density showed greater uncertainty compared to data with Gmax = 300 mT/m. In conclusion, the compartment size and volume fraction estimates resulting from diffusion measurements on a human scanner were validated against ground truth in a phantom mimicking human white matter, providing confidence that this method can yield accurate estimates of parameters in simplified but realistic microstructural environments. Our work also demonstrates the importance of a biologically analogous phantom that can be applied to validate a variety of diffusion microstructural imaging methods in human scanners and be used for standardization of diffusion MRI protocols for neuroimaging research.