The ability of Biosilicate® with two crystalline phases (BioS-2P) to drive osteoblast differentiation encourages the investigation of the cellular mechanisms involved in this process. Then, the aim of our study was to analyze the large-scale gene expression of osteoblasts grown on BioS-2P compared with Bioglass 45S5 (45S5). Osteoblasts differentiated from rat bone marrow mesenchymal stem cells were cultured under osteogenic conditions on BioS-2P, 45S5 and polystyrene (control). After 10 days, the expression of 23,794 genes was analyzed using mRNA Sequencing and the data were validated by real-time PCR. The BioS-2P exhibited 5 genes upregulated and 3 downregulated compared with 45S5. Compared with control, BioS-2P upregulated 15 and downregulated 11 genes, while 45S5 upregulated 25 and downregulated 21 genes. Eight genes were commonly upregulated and 4 downregulated by both bioactive glasses. In conclusion, our results demonstrated that bioactive glasses affect the gene expression profiling of osteoblasts. Most of the regulated genes by both BioS-2P and 45S5 are associated with the process of mineralization highlighting their osteostimulation property that is, at least in part, derived from the ability to modulate the intracellular machinery to promote osteoblast genotype expression. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 419-423, 2017.