AxCaliber is a novel NMR-based method for estimating axon diameter distribution from diffusion MRI data. This technique uses a model of water diffusion within restricted cylindrical axons to estimate axon diameter distribution within a nerve bundle. It can be combined with MRI to estimate axon diameter distribution within each voxel. The method was validated by comparing diameter distributions measured using NMR and histological techniques on sciatic and optic nerve tissue specimens. Axon diameter distribution measured in porcine spinal cord using MRI and histology were similar. Applications include longitudinal studies of nerve growth and diagnosing disorders affecting specific axon populations in the CNS and PNS. AxCaliber uses diffusion NMR and MRI data to estimate axon diameter distribution. Diffusion MRI provides unique information about white matter organization. Diffusion tensor imaging (DTI) is a leading method for analyzing neuronal fiber pathways. However, the Gaussian displacement distribution assumed by DTI is not adequate for describing water diffusion in intra-axonal spaces. AxCaliber extends the CHARMED framework by introducing the diameter distribution of restricted cylindrical axons as an unknown function to estimate using diffusion MR data with varying diffusion weighting and times. This approach allows for spectroscopic analysis of an entire specimen or MRI-based analysis within specific voxels. The method was validated using fixed porcine optic and sciatic nerve specimens and fixed porcine spinal cord specimens. AxCaliber NMR and MRI data were used to determine if spinal cord regions could be segmented based on similar diameter distributions. Histological analysis confirmed the accuracy of AxCaliber results. AxCaliber analysis involves modeling diffusion signals from hindered and restricted diffusion compartments. The hindered diffusion is modeled using the Stejskal-Tanner equation, while restricted diffusion is modeled using complex equations involving Bessel functions. The axon diameter distribution is modeled using a gamma function, with parameters fitted to diffusion data. AxCaliber imaging was used to segment spinal cord regions based on diameter distribution. Histological analysis confirmed the accuracy of AxCaliber results. The method provides a noninvasive way to measure axon diameter distribution, which is important for understanding white matter architecture, connectivity, and neuroanatomical changes in disorders. AxCaliber has limitations, including the need for multiple diffusion-weighted signals and the assumption that fibers run perpendicular to diffusion gradients. However, it provides unique information about tissue microstructure and can be used for in vivo studies. Future improvements include optimizing experimental design and incorporating nonparametric diameter distribution models. AxCaliber has the potential to enhance connectivity studies and improve segmentation of white matter fascicles.AxCaliber is a novel NMR-based method for estimating axon diameter distribution from diffusion MRI data. This technique uses a model of water diffusion within restricted cylindrical axons to estimate axon diameter distribution within a nerve bundle. It can be combined with MRI to estimate axon diameter distribution within each voxel. The method was validated by comparing diameter distributions measured using NMR and histological techniques on sciatic and optic nerve tissue specimens. Axon diameter distribution measured in porcine spinal cord using MRI and histology were similar. Applications include longitudinal studies of nerve growth and diagnosing disorders affecting specific axon populations in the CNS and PNS. AxCaliber uses diffusion NMR and MRI data to estimate axon diameter distribution. Diffusion MRI provides unique information about white matter organization. Diffusion tensor imaging (DTI) is a leading method for analyzing neuronal fiber pathways. However, the Gaussian displacement distribution assumed by DTI is not adequate for describing water diffusion in intra-axonal spaces. AxCaliber extends the CHARMED framework by introducing the diameter distribution of restricted cylindrical axons as an unknown function to estimate using diffusion MR data with varying diffusion weighting and times. This approach allows for spectroscopic analysis of an entire specimen or MRI-based analysis within specific voxels. The method was validated using fixed porcine optic and sciatic nerve specimens and fixed porcine spinal cord specimens. AxCaliber NMR and MRI data were used to determine if spinal cord regions could be segmented based on similar diameter distributions. Histological analysis confirmed the accuracy of AxCaliber results. AxCaliber analysis involves modeling diffusion signals from hindered and restricted diffusion compartments. The hindered diffusion is modeled using the Stejskal-Tanner equation, while restricted diffusion is modeled using complex equations involving Bessel functions. The axon diameter distribution is modeled using a gamma function, with parameters fitted to diffusion data. AxCaliber imaging was used to segment spinal cord regions based on diameter distribution. Histological analysis confirmed the accuracy of AxCaliber results. The method provides a noninvasive way to measure axon diameter distribution, which is important for understanding white matter architecture, connectivity, and neuroanatomical changes in disorders. AxCaliber has limitations, including the need for multiple diffusion-weighted signals and the assumption that fibers run perpendicular to diffusion gradients. However, it provides unique information about tissue microstructure and can be used for in vivo studies. Future improvements include optimizing experimental design and incorporating nonparametric diameter distribution models. AxCaliber has the potential to enhance connectivity studies and improve segmentation of white matter fascicles.