Sporosarcina pasteurii (S. pasteurii) is a kind of Gram-positive bacteria from soil. Various applications have been developed based on the efficient urease activity that can induce the precipitation of calcium carbonate. However, the metabolic mechanism related to biomineralization of S. pasteurii has not been clearly elucidated. Especially, there are few studies on the gene structure of urease, regulation mechanism of expression and associated metabolism, which play key roles in biomineralization. Nowadays, the uncontrollability and instability of biomineralization reactions in the applications of S. pasteurii root in the lack of research on urease metabolisms. Therefore, it is urgent to further reveal the gene information, expression regulation and related metabolism of urease in S. pasteurii.In this paper, we compared the growth and gene expression of S. pasteurii BNCC 337394 under four different culture conditions through high-throughput transcriptome analyses. The four medium conditions were: (1) control group with yeast extract of 20 g/L; (2) ammonium group with yeast extract of 20 g/L and ammonium chloride of 10 g/L; (3) urea group with yeast extract of 20 g/L and urea of 5.62 g/L; (4) ammonium Tris group with yeast extract of 20 g/L, ammonium chloride of 10 g/L and Tris of 15.75 g/L. Transcriptome data were analyzed by bioinformatics methods of differential gene expression analysis, GO enrichment analysis, KEGG enrichment analysis and operon prediction.The results showed that there were significant differences in the growth of S. pasteurii among the four conditions. The bacteria could not grow and reproduce normally in the control group. There was a growth delay in the ammonium Tris group. The growth rate and trend of the ammonium group and the urea group with the same nitrogen content were similar. A total of 3 090 genes were generated and expressed in S. pasteurii in the experiment. There were 1 152 differentially expressed genes (DEGs) between the ammonium group and the control group, of which 411 were up-regulated and 741 were down-regulated. There were 1 362 DEGs between the urea group and the control group, of which 736 were up-regulated and 626 were down-regulated. There were 803 DEGs between the ammonium group and the urea group, of which 279 were up-regulated and 524 were down-regulated. There were 794 DEGs between the ammonium group and the ammonium Tris group, of which 281 were up-regulated and 513 were down-regulated. Furthermore, it was found that the expression of urease was significantly enhanced in the control group which was short of a nitrogen source comparing with the ammonium group and the urea group.GO and KEGG analyses revealed that the control group needed to enhance its basal metabolism and express more flagellum in order to survive. The pathways of electron transfer activity and oxidative phosphorylation were different in the ammonium group and the urea group. It indicated that the synthesis of ATP was associated with the hydrolysis of urea in S. pasteurii. Meanwhile, ammonium stimulated the expression of urease and ATP synthase in the ammonium group which was more significant than that of the urea group. The results of the ammonium Tris group indicated that a stable and moderate alkaline pH environment favored a high level expression of urease. Finally, the double operon gene structure of urease was predicted based on the two key characteristics of gene expression similarity and gene spacing.