Fluid shear stress (FSS) is regarded as a predominant stimulus to bone cells and matrix under physiological conditions. The influence of FSS on mineralization of collagen is explored by exposure to an environment of constant FSS values less than 2.0 Pa. At the designated time points, the apatite/collagen composites were characterized by X-ray diffraction, calcium/phosphorus assay, differential scanning calorimetry, transmission electron microscopy, and selected area electron diffraction. The results show that FSS within a certain range, especially within 1.5 Pa, has a positive effect on collagen mineralization, as illustrated by the enhanced degree of collagen self-assembly, accelerated speed of amorphous calcium phosphate (ACP) formation and transition, and well-organized apatite structure and orientation. Under the condition of FSS, the size of ACP is also well controlled, and the minerals disperse inside collagen fibrils; this leads to intrafibrillar mineralization. These findings are helpful for understanding the mechanism of mineralization in natural bone tissue and deepen the knowledge of biomechanics of this process.