Osteoblast proliferation and migration are significantly involved in bone healing, regeneration and embryonic skeletal development. Recently, reports suggest anabolic properties of High Mobility Group Box Protein 1 (HMGB1) within the subsequent phase of tissue regeneration as well as HMGB1 has been shown to be highly expressed during bone fracture and regulates osteochondral ossification. In this study, the effect of various concentrations of Recombinant Human HMGB-1 (rhHMGB-1) (50, 100,150 and 200 µg/L) on the viability and proliferation of Pre-osteoblast cells MC3T3-E1 were examined over 24, 48 and 72 hours intervals in comparison with control, untreated cells. A cell count kit (CCk-8) was used to assess osteoblast viability and proliferation, results were quantified using a microplate reader and the viability %( survival rate) was calculated. All experiments were done in triplicate and outcomes were statistically analyzed. A direct positive impact of HMGB-1 on the viability and proliferation of osteoblast cells was in a dose dependent manner. Lower concentration of HMGB-1 (50 and 100 µg/L) showed a non-significance difference in the viability and proliferation of osteoblast cells over time, however, a higher concentration of HMGB-1 (150 and 200 µg/L) showed a significant increase in the proliferation and survival rate by 1.7 fold compared to control group in the 2^nd and 3^rd day of treatment. No significant difference was found between the 150 and 200 µg/L concentrations which suggested the appropriate dose will be 150 µg/L. Moreover, further in vivo and in vitro investigations will be required.