Abstract: Herein, commercial natural bovine tendon collagen (sponge-like) was employed as the raw material and processed into powder further via the cryo-grinding approach. Effects of pre-freezing duration on the molecular structure, physicochemical aptitudes and biological capabilities of the natural macromolecule were systematically probed. Results revealed that the polymer's triple-helix structure and intermolecular interactions were susceptible to the pre-freezing period. Specifically, gel electrophoresis experiment (sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE) and infrared spectrum (IR) unfolded that cryo-grinding within a short time, for instance, ten hours, will impose a particular influence on peptide chain structure of collagen molecules, which can be minimized after reinforced freezing for 24 h (COL24h). Circular dichroism (CD), atomic force microscope (AFM), and scanning electron microscope (SEM) confirmed collectively that the triple-helix structure of COL24h was the most intact, with a natural talent for fibrosis in vitro. More strikingly, as the solubility experiment revealed, the water solubility (4℃) of the ground ones soared with prolonged pre-freezing time; the solubility of COL24 was nearly four times larger than that of the control group (COL). In addition, thermal stability assay implied that cryo-grinding treatment also had an evident effect on the thermal enthalpy but not the denaturation temperature of the material; besides, with enhanced freezing duration, this impact was gradually reduced; rheological dissection proved that the overall viscosity of the collagen solution descended, exhibiting a better fluidity after grinding, and a cell test confirmed that COL24h had more substantial capacity to promote cell proliferation, shedding light on its potential amid medical cosmetology and regenerative medical materials.