In mechanotransduction studies, flow-induced shear stress (FSS) is often applied to two-dimensional (2D) cultured cells with a parallel-plate flow chamber (PPFC) due to its simple FSS estimation. However, cells behave differently under FSS inside a 3D scaffold (e.g. 10 mPa FSS was shown to induce osteogenesis of human mesenchymal stem cells (hMSC) in 3D but over 900 mPa was needed for 2D culture). Here, a simple in vitro biomimetic perfusion system using borosilicate glass capillary tubes has been developed to study the cellular behaviour under low-level FSS that mimics 3D culture. It has been shown that, compared to cells in the PPFC, hMSC in the capillary tubes had upregulated Runx-2 expression and osteogenic cytoskeleton actin network under 10 mPa FSS for 24 h. Also, an image analysis method based on Haralick texture measurement has been used to identify osteogenic actin network. The biomimetic perfusion system can be a valuable tool to study mechanotransduction in 3D for more clinical relevant tissue-engineering applications.