Tetracyclines (TCs) are a class of broad-spectrum antibiotics with diverse pharmacotherapeutic properties due to their various functional groups being attached to a common core structure. Beyond their antibacterial activity, TCs trigger pleiotropic effects on eukaryotic cells, including anti-inflammatory and potentially osteogenic capabilities. Consequently, TCs hold promise for repurposing in various clinical applications, including bone-related conditions. This study presents the first comprehensive comparison of the in vitro osteogenic potential of four TCs—tetracycline, doxycycline, minocycline, and sarecycline, within human mesenchymal stem cells. Cultures were characterized for metabolic activity, cell morphology and cytoskeleton organization, osteogenic gene expression, alkaline phosphatase (ALP) activity, and the activation of relevant signaling pathways. TCs stimulated actin remodeling processes, inducing morphological shifts consistent with osteogenic differentiation. Osteogenic gene expression and ALP activity supported the osteoinduction by TCs, demonstrating significant increases in ALP levels and the upregulation of RUNX2, SP7, and SPARC genes. Minocycline and sarecycline exhibited the most potent osteogenic induction, comparable to conventional osteogenic inducers. Signaling pathway analysis revealed that tetracycline and doxycycline activate the Wnt pathway, while minocycline and sarecycline upregulated Hedgehog signaling. Overall, the present findings suggest that TCs promote osteogenic differentiation through distinct pathways, making them promising candidates for targeted therapy in specific bone-related disorders.