Hydroxyapatite (HA) has been extensively explored for biomedical applications due to its advantages of facile synthesis and surface functions. Many studies have suggested that HA microparticles can induce osteogenic differentiation. However, how the size and shape of HA microparticles affected the differentiation response has not been elucidated. In this work, HA microparticles (HA whiskers and HA microspheres) with different sizes of 40, 50 and 80 μm were synthesized and their effects on the osteogenic differentiation of MC3T3-E1 preosteoblasts were investigated. All the HA microparticles showed cytocompatibility and did not show their influence on the proliferation at the studied concentrations. The osteogenic differentiation of MC3T3-E1 preosteoblasts was dependent on the size and shape of HA. Sphere-50 and sphere-80 significantly increased the alkaline phosphatase (ALP) activity, calcium deposition and expression of Runx2 and collagen I, while whisker-40, whisker-50 and whisker-80 reduced the ALP activity and calcium deposition. The results indicated that the size and shape of HA microparticles have an influence on the osteogenic differentiation of MC3T3-E1 preosteoblasts.