Objective To explore the effects of dynamic axial compressive strain on the mRNA expression of bone formation related-genes in osteoblasts seeded in 3D silk fibroin scaffolds. Methods In the experimental group, MC3T3-E1 cells were seeded in 3D scaffolds and then subjected to dynamic axial compressive strain (at amplitude of 5% and frequency of 1 Hz, 30 min/day for 20 days), while in the control group, MC3T3-E1 cells were cultured statically without any mechanical stimulation. The gene expression of alkaline phosphatase (ALP), collagenⅠ(COL-Ⅰ), runt-related transcription factor 2 (Runx2), osterix (Osx), osteocalcin (OCN) was detected by real-time PCR. Results Under cyclic axial compressive strain, the Runx2, Osx and COLⅠmRNA levels increased by 280%, 68.9% and 79.6%, respectively, while the ALP and OC mRNA levels also up-regulated by 10.7% and 26.9%, respectively. There were significant differences in mRNA expression of osteogenesis-related genes between the experimental group and control group (P＜0.05). Conclusions Under the stimulation of cyclic axial compressive strain, the osteogenic differentiation of osteoblasts seeded in the silk fibroin scaffolds is promoted, with a significant up-regulation in the mRNA expression of COLⅠ, Runx2, Osx and OCN, which suggests that the stimulation of compressive stress at physiologic magnitude could be one of important mechanisms in promoting fracture healing. This research finding will be important for the clinic application of mechanical stimuli-mediated cell therapy for bone defection.