Objective: To acquire recombinant nematode anticoagulant peptide (NAP) with high anticoagulant activity to lay a foundation for the development of novel anticoagulant agent. Methods: Pichia pastoris GS115 strain was transformed with recombinant yeast expression vector pPIC3.5K-rNAP. Expression of rNAP was induced with methanol after the identification of positive strains. NAP expressed in the collected yeast culture supernatant was confirmed with SDS-PAGE and Western blot. The biological activity of the products was validated with PT (prothrombin time), INR (international normalized ratio) and APTT (activated partial thromboplastin time), respectively. Results: The yeast strains stably express NAP were identified. The rNAP was secreted into culture supernatant with a molecular weight of about 10 kD due to glycosylation, which is a little bigger than that predicted (8.7kD). The anticoagulant efficiency of rNAP was confirmed with the in vitro assays. Conclusion: The recombinant nematode anticoagulant peptide with high biological activity was successfully expressed in Pichia pastoris and can be used in the future development of novel anticoagulant agent.