Osteocytes play a pivotal role in the regulation of skeletal mass. Osteocyte processes are thought to sense the flow of interstitial fluid that is driven through the osteocyte canaliculi by mechanical stimuli placed upon bone, but how this flow elicits a cellular response is virtually unknown. Modern theoretical models assume that osteocyte canaliculi contain ultrastructural features that amplify the fluid flow-derived mechanical signal. Unfortunately the calcified bone matrix has considerably hampered studies on the osteocyte process within its canaliculus. Using one of the few ultra high voltage electron microscopes (UHVEM) available worldwide, we applied UHVEM tomography at 2 MeV to reconstruct unique three-dimensional images of osteocyte canaliculi in 1 μm sections of human bone. A realistic three-dimensional image-based model of a single canaliculus was constructed, and the fluid dynamics of a Newtonian fluid flow within the canaliculus was analyzed. We created virtual 2.2 nm thick sections through a canaliculus and found that traditional TEM techniques create a false impression that osteocyte processes are directly attached to the canalicular wall. The canalicular wall had a highly irregular surface and contained protruding axisymmetric structures similar in size and shape to collagen fibrils. We also found that the microscopic surface roughness of the canalicular wall strongly influenced the fluid flow profiles, whereby highly inhomogeneous flow patterns emerged. These inhomogeneous flow patterns may induce deformation of cytoskeletal elements in the osteocyte process, thereby amplifying mechanical signals. Based on these observations, new and realistic models can be developed that will significantly enhance our understanding of the process of mechanotransduction in bone.