Background <p>Fibrosarcoma is an aggressive soft tissue malignancy with limited therapeutic options, highlighting the need for novel treatment strategies. Nafamostat mesylate, a clinically approved serine protease inhibitor, has demonstrated anticancer effects in various tumor types, yet its impact on fibrosarcoma remains unexplored. This study aimed to investigate the cytotoxic, antimigratory, pro-apoptotic, and anti-invasive effects of nafamostat mesylate in human HT1080 fibrosarcoma cells.</p> Methods <p>HT1080 cells were treated with varying concentrations of nafamostat mesylate. Cell viability was assessed by MTT assay, migration by wound healing assay, and cell cycle distribution by flow cytometry. Apoptosis induction was evaluated using Annexin V binding assay, multicaspase activity, and mitochondrial membrane potential analysis. Additionally, mRNA expression levels of matrix metalloproteinases (MMPs), MMP-2 and MMP-9, were quantified by qRT-PCR.</p> Results <p>Nafamostat mesylate reduced HT1080 cell viability in a dose- and time-dependent manner and induced G2/M cell cycle arrest, indicating disruption of mitotic progression. Migration assays demonstrated suppression of cell motility. Apoptosis was confirmed through increased Annexin V positivity, elevated caspase activity, and mitochondrial depolarization, supporting caspase-dependent, mitochondria-mediated cell death. Furthermore, nafamostat mesylate treatment significantly downregulated MMP-2 and MMP-9 mRNA expression, suggesting inhibition of key enzymes responsible for extracellular matrix (ECM) degradation, invasion and metastasis.</p> Conclusions <p>This study provides the first evidence that nafamostat mesylate exerts multifaceted anticancer effects in HT1080 fibrosarcoma cells, targeting proliferation, migration, apoptosis, and invasion. These findings support the potential repurposing of nafamostat mesylate as a therapeutic agent for fibrosarcoma and warrant further preclinical investigations to evaluate its translational applicability.</p>