It has been observed that certain strains of the bacterium Gluconacetobacter xylinus can produce sphere-like cellulose particles (SCP) under orbital shaking cultivation. These unique particles may have broad applications in materials science, especially in the biomedical field. The mechanism behind SCP formation and SCP biocompatibility, however, remain unknown. In this study, several factors potentially involved in the formation of SCP have been examined including the composition of initial inoculums, inoculum volume, initial media glucose concentration, and temperature. The results revealed that cellulose fibers supposedly existing in the initial inoculums did not relate to the initiation of cellulose spherical structure. Increased inoculum volume reduced the number of SCP, and different initial glucose concentrations impacted the mean of approximate diameters of SCP, while the number of SCP remained unchanged under different initial glucose concentrations. Additionally, the formation process of SCP has been clearly identified in this study by lowering the culture temperature. Furthermore, rapid attachment and extension of human osteoblast cells grown on SCP demonstrated their good biocompatibility and the potential use of this kind of materials for biomedical applications.