Background Osteogenic differentiation is a crucial process in which bone marrow mesenchymal stem cells (BMSCs) differentiate into osteoblasts, involving the regulation of multiple genes and signaling pathways. The TSC22D3 gene plays an important role in various biological processes (BPs), but its specific function in osteogenic differentiation remains unclear. This study aims to explore the regulatory role of the TSC22D3 gene in osteogenic differentiation and its molecular mechanisms. Methods By analyzing microarray datasets (GSE12266, GSE18043, and GSE80614), the limma package was used to screen for differentially expressed genes (DEGs). Combined with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, key genes and signaling pathways related to osteogenic differentiation were identified. Further, through protein–protein interaction (PPI) network analysis and the Finding Regulatory Elements by Differential Expression and Network‐Based Statistical Analysis (FRIEND) method, TSC22D3 was screened out as a core hub gene. For experimental validation, the bioinformatics analysis results were intersected with the transcriptome sequencing data from our research group to further confirm the core molecules. Lentivirus‐mediated interference technology was used to downregulate and overexpress TSC22D3 expression, and the impact of TSC22D3 on osteogenic differentiation was assessed through RT‐qPCR, Western blotting, alkaline phosphatase (ALP) staining, phalloidin staining, and calcium deposition assays. Results TSC22D3 is significantly upregulated during osteogenic differentiation; its downregulation can lead to reduced expression of osteogenic differentiation marker genes (such as runt‐related transcription factor 2 [Runx2], osterix [OSX], osteocalcin [OCN], and osteopontin [OPN]), as well as a significant decrease in ALP activity and calcium deposition. GO and KEGG analyses indicate that TSC22D3 is closely associated with pathways including the cell cycle, cytoskeleton, and WNT signaling. Furthermore, Gene Set Enrichment Analysis (GSEA) analysis has further revealed the potential regulatory mechanism of TSC22D3 in osteogenic differentiation. Rescue experiments have confirmed that TSC22D3 can promote the osteogenic differentiation of BMSCs and induce the rearrangement of cytoskeletal structure. Conclusion This study reveals that TSC22D3 is essential for osteogenic differentiation. Its upregulation promotes osteogenic marker expression, ALP activity, and calcium deposition, while its downregulation inhibits these processes. TSC22D3 affects cytoskeletal rearrangement during osteogenesis.