A key problem of how to distinguish between biotic and abiotic carbonate minerals in nature and/or in the laboratory has confused researchers for some time. Although numerous studies have been performed to explore the formation mechanism and the unique characteristics of biotic minerals, the issue needs to be studied further. Here, biotic calcite precipitation was induced by the microorganism Bacillus cereus MRR2 (GenBank KY810857), while organogenic and inorganogenic calcite was investigated in parallel. The results indicate that carbonic anhydrase (CA, a common enzyme of bacteria) promoted carbon dioxide hydration to release HCO3− and CO32− in the process of calcite precipitation; the pH increased from 7.2 to 8.9 as a result of the combination effect of the released ammonia by B. cereus MRR2 and the HCO3− and CO32− ions originating from the CA reaction. Glutamic acid is the most abundant amino acid in the extracellular polymeric substances of the bacteria, and its free carboxyl ions with a large number of negative charges in the alkaline environment facilitated the adsorption of calcium ions. The biotic calcite has a series of unique characteristics different from the organogenic and inorganogenic calcites, such as particular morphologies, P element composition and a variety of organic functional groups. Intracellular amorphous nanospheres without any crystal structure are also recorded. Stable carbon isotope analyses show that the biotic calcite (−20.9‰) has more negative δ13C values than the organogenic calcite (−15.6‰) and inorganogenic calcite (−11.7‰), indicating that microbial activity strongly affected the carbon isotope composition of biotic calcite. More importantly, the crystallinity and activation energy of the biotic calcite are clearly higher than those of organogenic calcite, suggesting that the thermal stability of biotic calcite is much higher. Thus, this study provides important insights into understanding the mechanisms of microbial biomineralization and the processes of biotic calcite formation, especially the roles microbes played in mineral nucleation and growth, as well as the unique characteristics of the resulting minerals. This study may provide useful evidence for further recognizing biotic and abiotic calcite in the geological record. The model of biotic, organogenic and inorganogenic calcite. Display omitted •The pH increase is caused by the combination effect of ammonia and CA.•Biotic calcite has higher thermal stability than abiotic calcite.•Biotic calcite has more negative δ13C values than abiotic calcite.•Glutamic acid is the most abundant amino acid in EPS.•The intracellular amorphous nanospheres are found.