Abstract: Objective To study the effect of recombinant adenovirus-mediated NELL1 on proliferation and differentiation of osteoblasts（MC3T3E1） and expression of osteocalcin（OCN） and osteopontin（OPN） in mice. Methods Expression of recombinant adenovirus-transfected green fluorescence protein（GFP） and NELL1 was identified with immunofluorescence staining.MC3T3E1 transfected with adenovirus-mediated NELL1 were observed under fluorescent microscope and divided into control group,NELL1 group,and GFP group.Growth curve was plotted for MC3T3E1 with a CCK-8 kit.The number of calcium nodules was calculated with alizarin red staining.Expression of osteoblast master genes（OCN and OPN） was detected by real-time PCR. Results The immunofluorescence showed that the bone marrow matrix cells expressed both GFP and NELL1 72h after transfected with adenovirus-mediated NELL1.At the best multiplicity of infection（MOI）=200pfu/cell,the adenovirus-mediated NELL1 and GFP had no significant effect on the proliferation of MC3T3E1.The number of calcium nodules was significantly greater in NELL1 group than in control group and GFP group（7±2 vs 2±1 and 2±1,P＜0.05）.The expression levels of OCN and OPN were 3.7-fold and 5.1-fold higher in NELL1 group than in control group（P＜0.05）. Conclusion Adenovirus-mediated NELL1 can effectively promote the differentiation of osteoblasts with no significant toxicity to their proliferation.