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Degradable phosphate glasses have shown favorable properties for tissue engineering. By changing the composition of the glasses, the degradation rate, and ion release are controllable. Zinc oxide can function as a glass network modifier and has been shown to play a positive role in bone formation. Also, phosphate glasses can easily be processed into microspheres, which can be used as microcarriers. This study aims to develop zinc phosphate glasses microspheres and explore the optimized size and composition for applications in bone tissue engineering. Zinc–titanium–calcium–sodium phosphate glasses with 0, 1, 3, 5, or 10 mol % zinc oxide were prepared and processed into microspheres. The smaller microspheres ranged in size from 50 to 106 μm, while the larger ones ranged from 106 to 150 μm. The characteristics of glasses were examined. The osteoblastic cell line MC3T3‐E1 was cultured on the surface of microspheres and the cell viability was examined. To evaluate osteogenic differentiation, Alizarin Red S staining, quantitative reverse transcription polymerase chain reaction, and western blot analysis were performed after 14 days. Different sizes of zinc phosphate glass microspheres were successfully made. The glass microspheres with <10 mol % zinc oxide were able to support the adhesion and proliferation of MC3T3‐E1 cell lines. The relative gene expression of BMP2 was significantly upregulated in the smaller glass microspheres containing 3 mol % zinc oxide (26‐fold, p <.001) and both sizes of microspheres containing 5 mol % zinc oxide (smaller: 27‐fold, p <.001; larger: 35‐fold, p <.001). Additionally, cluster formation was observed in glass microspheres after 14 days, and the mineralization of MC3T3‐E1 cell lines was promoted. Based on these findings, the glass microspheres containing 3–5 mol % of zinc oxide can promote osteogenic differentiation for MC3T3‐E1 cells. [ABSTRACT FROM AUTHOR]