Objective To observe the effect of dynamic mechanical loading on the proliferation, differentiation, and specific gene expression of MC3T3-E1 cells that on three-dimensional (3D) biomimetic composite scaffolds prepared by low temperature 3D printing technology combined with freeze-drying. Methods The silk fibroin, collagen type Ⅰ, and nano-hydroxyapatite (HA) were mixed at a mass ratio of 3∶9∶2 and were used to prepare the 3D biomimetic composite scaffolds via low temperature 3D printing technology combined with freeze-drying. General morphology of 3D biomimetic composite scaffold was observed. Micro-CT was used to observe the pore size and porosity of the scaffolds, and the water swelling rate, stress, strain, and elastic modulus were measured. Then, the MC3T3-E1 cells were seeded on the 3D biomimetic composite scaffolds and the cell-scaffold composites were randomly divided into 2 groups. The experimental group was subjected to dynamic mechanical loading (3 500 με, 1 Hz, 15 minutes per day); the control group was not subjected to loading treatment. After 7 days and 14 days, the cell-scaffold composites of 2 groups were harvested to observe the growth of cells on the scaffolds by HE staining and scanning electron microscope. And the gene and protein expressions of collagen type Ⅰ, BMP-2, and osteocalcin (OCN) were measured by real-time fluorescent quantitative PCR and Western blot. Results The 3D biomimetic composite scaffold was a white cubic grid. Micro-CT detection showed the pore network structure in the scaffold material with good pore connectivity. The diameters of large pore and micro-aperture were (506.37±18.63) μm and (62.14±17.35) μm, respectively. The porosity was 97.70%±1.37%, and the water absorption swelling rate was 1 341.97%±64.41%. Mechanical tests showed that the compression displacement of the scaffold was (0.376±0.004) mm, the compressive stress was (0.016±0.002) MPa, and the elastic modulus was (162.418±18.754) kPa when the scaffold was compressed to 10%. At 7 days and 14 days, HE staining and scanning electron microscope observation showed that the cells grew inside the scaffold, mainly distributed around the scaffold pore wall. The cells in experimental group were more than control group, and the cells morphology changed from shuttle to flat. There was no significant difference in the cell counting between 2 groups at 14 days after 200-fold microscopy (t=–2.024, P=0.080), but significant differences were found between 2 groups at different time points under different magnifications (P<0.05). Real-time fluorescent quantitative PCR showed that the mRNA relative expressions of collagen type Ⅰ and OCN in experimental group were significantly higher than those in control group at 7 and 14 days (P<0.05). However, the mRNA relative expression of BMP-2 showing no significant difference between 2 groups (P>0.05). The protein relative expressions of collagen type Ⅰ, BMP-2, and OCN in experimental group were significantly higher than those in control group at 7 and 14 days (P<0.05). Conclusion After dynamic mechanical loading, the expressions of BMP-2, collagen type Ⅰ, and OCN in MC3T3-E1 cells inoculated into 3D biomimetic composite scaffolds are significantly up-regulated, indicating that appropriate mechanical loads favor osteoblast differentiation of MC3T3-E1 cells.