Abstract: A large number of microbes can mediate the formation of carbonate spherulite. Tailing the process of carbonate spherulite formation is the key to understand the mechanism. Using optic microscope and electron microscope, the whole process of carbonate spherulite formation under the mediation of Citrobacter freundii was traced in this study. The results showed that nanometer crystals formed firstly, and later gradually aggregated which were embedded by organics. Finally, spherulite structure formed with radiating inner structure and prismatic rhombic surface texture. During this process, several intermediate structures occurred, mainly with the morphologies of flowers, dumbbells, cauliflowers, spindles and crosses. The structural evolution offers insight into the mechanism of carbonate spherulite formation. It might be considered as the microbial signature in carbonate sediments and rocks. Because previous studies have been rarely conducted for the carbonate precipitation by Citrobacter freundii,this study will provide more information to microbial-mediated carbonate spherulite formation.