Osteocyte shear stresses and cytoskeleton deformation resulting from bone interstitial fluid flow in the lacunar-canalicular porosity have been proposed to be involved in bone’s mechanotransduction mechanism. However, the fluid flow properties that regulate bone’s adaptive response are poorly understood. We first present a two-step analytical approach to determine the degree of anisotropy of the permeability of the lacunar-canalicular porosity in bone. In the first step, we estimate the total number of canaliculi emanating from each osteocyte lacuna based on published measurements for several species (chick, rabbit, cow, horse, dog, and man). In the second step, we determine the local three-dimensional permeability of the lacunar-canalicular porosity for these species by adapting a previously developed microstructural model. Permeability coefficients for the lacunar-canalicular porosity were found to exhibit local orthotropic symmetry for all species examined. We next designed experiments that provide a complete three-dimensional characterization of bone’s microstructure to confirm and quantify the essential bone parameters used in our model. Measurements of the osteocyte lacuna axes length, the volumetric lacunae density, the number of canaliculi emanating per lacuna, the number of canaliculi intersected by the bone territory assigned to each lacuna, and the canaliculus annulus size were obtained in rat bone using confocal laser scanning microscopy. The experimentally measured number of canaliculi emanating per osteocyte lacuna falls within the range of values estimated in our theoretical study. One aspect evident in rat bone that was not considered in the theoretical study was that many canaliculi emanating from the osteocyte lacuna exhibited branching. The permeability coefficients calculated using the rat confocal lacunar-canalicular measurements were found to represent local orthotropic symmetry of bone permeability, although the degree of orthotropy was different from our previous theoretical calculations mostly due to the branching of canaliculi. The local lacunar-canalicular permeability coefficients presented here will provide an important material property dataset that can be used to build poroelastic finite element models of bone. These finite element models will also include the vascular porosity, thus providing the most realistic models to date to solve for load-induced interstitial fluid pressure and velocity fields in complex bone geometries.