A novel high-throughput strategy was developed to determine the calcium precipitation activity (CPA) of mineralization bacteria used for self-healing of concrete cracks. A bacterial strain designated as H4 with the highest CPA of 94.8 % was screened and identified as a Bacillus species based on 16S rDNA sequence and phylogenetic tree analysis. Furthermore, the effects of certain influential factors on the microbial calcium precipitation process of H4 were evaluated. The results showed that lactate and nitrate are the best carbon and nitrogen sources, with optimal concentrations of approximately 25 and 18 mM, respectively. The H4 strain is able to maintain a high CPA in the pH range of 9.5–11.0, and a suitable initial spore concentration is 4.0 × 10 7 spores/ml. Moreover, an ambient Ca 2+ concentration greater than 60 mM resulted in a serious adverse impact not only on the CPA but also on the growth of H4, suggesting that the maintenance of the Ca 2+ concentration at a low level is necessary for microbial self-healing of concrete cracks.