Background: Protease nexin-1 (PN-1) is a member of the serine protease inhibitor (Serpin)-family, with thrombin as its main target. Current polyclonal and monoclonal antibodies against PN-1 frequently cross-react with Plasminogen activator inhibitor-1 (PAI-1), a structurally and functionally homologous Serpin. Objectives: Here, we aimed to develop inhibitory single-domain antibodies (VHHs) that show specific binding to both human (hPN-1) and murine (mPN-1) PN-1. Methods: PN-1-binding VHHs were isolated via phage-display using llama-derived or synthetic VHH-libraries. Following bacterial expression, purified VHHs were analyzed in binding and activity assays. Results and Conclusions: By using a llama-derived library, 2 PN-1 specific VHHs were obtained (KB-PN1-01 & KB-PN1-02). Despite their specificity, none displayed inhibitory activity towards hPN-1 or mPN-1. From the synthetic library, 4 VHHs (H12, B11, F06, A08) could be isolated that combined efficient binding to both hPN-1 and mPN-1 with negligible binding to PAI-1. Of these, B11, F06 and A08 were able to fully restore thrombin activity by blocking PN-1. As monovalent VHH, IC50-values for hPN-1 were 50±10 nM, 290±30 and 960±390 nM, for B11, F06 and A08, respectively, and 1580±240 nM, 560±130 nM and 2880±770 nM for mPN-1. The inhibitory potential was improved 4- to 7-fold when bivalent VHHs were engineered. Importantly, all VHHs could block PN-1 activity in plasma as well as PN-1 released from activated platelets, one of the main sources of PN-1 during hemostasis. In conclusion, we report the generation of inhibitory anti-PN-1 antibodies using a specific approach to avoid cross-reactivity with the homologous Serpin PAI-1.