Abstract This study was designed to investigate the effect of surfactin C, which is derived from Bacillus subtilis, on platelet aggregation and homotypic leucocyte aggregation. Surfactin C strongly and dose-dependently inhibited platelet aggregation, which was stimulated both by thrombin (0.1 U mL−1), a potent agonist that activates the G protein-coupled protease receptor, and by collagen (5 μg mL−1), a potent ligand that activates αIIbβ3 with IC50 values (concentration inhibiting platelet aggregation by 50%) of 10.9 and 17.0 μM, respectively. Moreover, surfactin C significantly suppressed the intracellular Ca2+ mobilization in thrombin-activated platelets. Surfactin C, however, did not affect various integrin-mediated U937 cell aggregation, implying that the anti-platelet activity of surfactin C was not due to its detergent effect but by its action on the downstream signalling pathway. Therefore, the results suggest that surfactin C may have a beneficial therapeutic effect on aberrant platelet aggregation-mediated cardiovascular diseases.