Background and Purpose: Mesenchymal stem cells (MSCs) are widely utilized in regenerative medicine due to their multipotency and immunomodulatory properties. Compared to conventional two-dimensional (2D) monolayer cultures, three-dimensional (3D) spheroid cultures better mimic the in vivo microenvironment, influencing the metabolic activity and therapeutic efficacy of MSCs. This study aims to evaluate how 2D and 3D culture conditions affect the behavior, proliferation, and functional properties of MSCs. Experimental Approach: Metabolomic and transcriptomic analyses were conducted on MSCs cultured under both 2D and 3D conditions. To assess metabolic differences between 2D and 3D cultured MSCs, polar metabolites were extracted and analyzed using ¹H-NMR spectroscopy. The data was processed with Chenomx and subjected to multivariate statistical analysis. For transcriptomic analysis, RNA sequencing was performed, followed by differential gene expression and gene set enrichment analysis. Key Results: The findings reveal that MSCs in 3D spheroids exhibit reduced proliferation, enhanced stemness, and distinct metabolic adaptations, including increased glycolysis and altered nutrient metabolism. Additionally, genes associated with ribosome biogenesis and cell cycle progression were downregulated in 3D MSCs. These changes promote a more quiescent state, favoring its applications on tissue repair and immune modulation. Conclusion and Implications: Understanding these metabolic adaptations offers valuable insights for optimizing culture conditions, improving MSC-based therapies, and identifying novel therapeutic targets and biomarkers.