Objective To establish a New Zealand rabbit bone marrow mesenchymal stem cells (BMSCs)-gentamicin-calcium alginate 3D sustained-release culture system and to study the growth and differentiation of BMSCs. Methods BMSCs-calcium alginate 3D culture system (W group) and BMSCs-gentamicin-calcium alginate 3D sustained-release culture system (U group) were constructed and were cultured with HG-DMEM (15% FBS, 10 ng/mL TGF-β₁) under saturated humidity, 37℃ and at 5% CO₂ , with the culture medium changed on a daily basis, and the cell morphology and microsphere morphology changes were observed. H-E staining, toluidine blue staining and type Ⅱ collagen staining were performed for the microspheres on week 2, 4, and 6. Results Cell clusters were formed locally in the two groups after the 3D microspheres were cultured for 10 days. A large number of cell clusters were formed after 21 days, and BMSCs maintained a spherical or approximate spherical shape. There was no significant difference in cell proliferation or growth between the two groups (P>0.05). After a 2-week culture, toluidine blue staining of microspheres showed positive staining in both groups, but with no obvious extracellular matrix formation, and staining for collagen type Ⅱ antibody was weakly positive. After a 4-week culture, toluidine blue staining was obvious in the periphery of the cell microspheres in both groups, but the staining was unapparent in the center; extracellular matrix around the cell clusters had less blue colored substance, and the central cell clusters had more mauve substance; collagen type Ⅱ staining was strongly positive in both groups. Conclusion Local sustained-release of appropriate amount of gentamicin has no noticeable effect on the growth and transformation of BMSCs while reaching the minimum inhibitory concentration. The influence of Gentamicin on ultrastructure of BMSCs and chondrocytes remains to be further investigated.