BACKGROUND: Levofloxacin, a widely used fluoroquinolone antibiotic, has been linked to musculoskeletal complications. However, its impact on bone marrow mesenchymal stem cells (BMSCs), which are vital for tissue repair and regeneration, remains poorly understood. AIM: This study aims to examine the impact on rat BMSCs following therapy with levofloxacin. METHODS: Rat BMSCs were exposed to various doses of levofloxacin (0, 14, 28, 56, 112, and 224 μM) to assess its possible cytotoxic impact on these stem cells. Cell viability was assessed using the MTT assay to evaluate the cytotoxic effects of levofloxacin. Cell apoptosis was calculated with Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining, along with the expression levels of matrix metalloproteinase-3 (MMP-3), MMP-13, collagen type I alpha 1 (Col1A1), tissue inhibitor of metalloproteinase-1 (TIMP-1), and TIMP-3 messenger RNA (mRNA), which were assessed using RT-PCR. An apoptotic marker, caspase-3, was detected by immunocytochemical analysis. RESULTS: In certain concentrations (0-224 μM), as the concentration of levofloxacin increased, the number of apoptotic cells increased. The results demonstrated that levofloxacin significantly upregulated the mRNA levels of MMP-3 as well as MMP-13 in a dose-related manner, simultaneously downregulating TIMP-1 expression. In contrast, the expression of TIMP-3 and Col1A1 remained unaffected. In addition, the expression of caspase-3 was substantially elevated by levofloxacin in a concentration-related manner, between 28 μM and 224 μM, as indicated by immunocytochemistry. CONCLUSION: These findings provide evidence that levofloxacin exerts cytotoxicity on BMSCs, shown by increased apoptosis and a reduction in extracellular matrix components, highlighting a potential adverse impact of levofloxacin. Additionally, this cytotoxic effect may negatively affect fracture healing and impair the regenerative capacity of BMSCs.