Mechanical cues play an important role in bone regeneration and affect production and secretion dynamics of growth factors (GFs) involved in osteogenesis. The in vitro models for investigating the mechanoresponsiveness of the involvement of GFs in osteogenesis are limited to two-dimensional monolayer cell culture studies, which do not effectively embody the physiological interactions with the neighboring cells of different types and the interactions with a natural extracellular matrix. Natural bone formation is a complex process that necessitates the contribution of multiple cell types, physical and chemical cues in a three-dimensional (3D) setting. There is a need for in vitro models that represent the physiological diversity and characteristics of bone formation to realistically study the effects of mechanical cues on this process. In vitro cultures of marrow explants inherently ossify and they embody the multicellular and 3D nature of osteogenesis. Therefore, the aim of this study was to assess the mechanoresponsiveness of the scaffold-free, multicellular, and 3D model of osteogenesis based on inherent marrow ossification and to investigate the effects of mechanical loading on the osteoinductive GF production dynamics of this model. These aims were achieved by (1) culturing rat bone marrow explants for 28 days under basal conditions that facilitate inherent ossification, (2) employing mechanical stimulation (compressive loading) between days 12 and 26, and (3) quantifying the final ossified volume (OV) and the production levels of bone morphogenetic protein-2, vascular endothelial growth factor, insulin-like growth factor-1, and transforming growth factor-β1. The results showed that the final OV of the marrow explants increased by about fourfold in mechanically stimulated samples. Further, mechanical stimulation sustained the production level of vascular endothelial growth factor (starting day 21), which otherwise declined temporally under static conditions. The production levels of insulin-like growth factor-1 and transforming growth factor-β1 were enhanced under the effect of loading after day 21. In addition, significant correlations were observed between the final OV and the levels of GFs analyzed. In conclusion, this study demonstrates that the scaffold-free, multicellular, and 3D model of bone formation based on inherent ossification of marrow tissue is mechanoresponsive and mechanical loading improves in vitro osteogenesis in this model with sustaining or enhancing osteoinductive GF production levels, which otherwise would decline with increasing time.