Bone-related diseases such as osteoporosis pose a significant health economic burden to countries around the world and, because current treatments are insufficient, more effective therapies are desperately needed. This study explored the potential of Lactobacillus paracasei L30 extract to influence the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs). Our results showed that L30 extract significantly enhanced the expression of osteogenic markers in hBM-MSCs, including alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), and collagen type I alpha 1 (COL1A1). Mechanistic studies revealed that L30 extract activated the p38 MAPK and AKT signaling pathways, leading to phosphorylation of Glycogen synthase kinase-3 beta (GSK3β) and subsequent nuclear translocation of β-catenin. Conversely, inhibition of p38 MAPK, AKT, or knockdown of β-catenin significantly attenuated the osteogenic effects of L30 extract on hBM-MSCs. Furthermore, we found that L30 extract promoted osteogenic differentiation in primary osteoblast precursors isolated from mouse calvaria and enhances bone formation in ex vivo calvarial organ cultures. Therefore, the application of Lactobacillus paracasei L30 extract in such contexts could serve as a therapeutic approach for promoting bone formation. Collectively, our findings suggest a novel approach for the clinical management of bone-related disorders, with possible applications for treating diseases such as osteoporosis.