We have previously identified and characterized Drosophila hemolectin (Hml) in the conditioned medium of a Drosophila cell line. The deduced amino acids sequence contains a number of domains conserved in human von Willebrand factor, coagulation factor V/VIII, and complement factors. To characterize Hml localization and function, we have established two transgenic lines (hml-GAL4 and UAS-hmlRNAi). By crossing hml-GAL4 with UAS-eGFP, we observed that Hml is specifically expressed in embryonic and in larval hemocytes. We determined that Hml is expressed in a subpopulation of plasmatocytes and crystal cells but not in lamellocytes. hml RNAi larvae are viable and do not display obvious developmental defects under normal conditions. However, they show a bleeding defect upon injury. We confirmed that the failure to seal a wound is not due to a defect in melanin production or in phenoloxidase activity. The expression of antimicrobial peptides was not significantly affected on hml RNAi adults. Altogether, our data strongly suggest that Hml is involved in hemostasis and/or coagulation in Drosophila larvae.