The aim of the present investigation was to study the effects of mechanical strain on the orthopedic biomaterial Ti-6Al-4V-osteoblast interface, using an in vitro model. Homogeneous strain was applied to Human Bone Marrow derived Osteoblasts (HBMDOs) cultured on Ti-6Al-4V, at levels which are considered physiological, by a four-point bending mechanostimulatory system. A simple model for the estimation of maximum hydrodynamic shear stresses developed on cell culture layer and induced by nutrient medium flow during mechanical loading, as a function of the geometry of the culture plate and the load characteristics, is proposed. Shear stresses were lower than those which can elicit cell response. Mechanical loading was found that contributes to the regulation of osteoblast differentiation by influencing the expression of the osteoblast-specific transcription factor Cbfa1, both at the mRNA and protein level, and also the osteocalcin expression, whereas osteopontin gene expression was unaffected by mechanical loading at all experimental conditions.