Mechanical loading factors at the bone-implant interface are critical for the osseointegration and clinical success of the implant. 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 physiological levels (strain magnitudes 500 microstrain (microepsilon) and 1000 microepsilon, at frequencies of load application 0.5 Hz and 1 Hz), by a mechanostimulatory system, based on the principle of four-point bending. Semi-quantitative reverse transcription-polymerase chain reaction (sqRT-PCR) was used to determine the mRNA expression of Cbfa1 and osteocalcin at different loading conditions. The release of growth factors as a response to stretch was also investigated by transferring stretch-conditioned media to nonstretched cells and by measuring their effect on the regulation of DNA synthesis. Mechanical loading was found to contribute to the regulation of osteoblast differentiation by influencing the level of the osteoblast-specific transcription factor Cbfa1, both at the mRNA and protein level, and also the level of osteocalcin, which is regarded as the most osteoblast-specific gene. Both genes were differentially expressed shortly after the application of different mechanical stimuli, in terms of strain frequency, magnitude, and time interval. Media conditioned from mechanically stressed HBMDOs stimulate DNA synthesis more intensely compared to media conditioned from unstressed control cultures, indicating that mechanical strain induces the release of a mitogenic potential that regulates cell proliferation.