Anticoagulant activity was detected in fractions of a reversed phase-high-performance liquid chromatography (RP-HPLC) of salivary gland lysate from Simulium vittatum Zetterstedt. Using a plasma recalcification time assay, these fractions did not inhibit factor Xa or thrombin. HPLC-purified fractions showing the anticoagulant property were pooled and examined using the activated partial thromboplastin time test conducted on normal plasma and plasmas deficient in factors V, VIII, IX, XI, and XII. The anticoagulant prolonged the clotting time of all the plasmas, except plasma deficient in factor V. The detection of antifactor V activity, together with other anticoagulants reported from Simulium spp. indicates a feeding strategy that targets enzymes in the terminal portion of the coagulation cascade.