Coagulation factor XII (FXII) is a key initiator of the contact pathway, which contributes to inflammatory pathways. FXII circulates as a zymogen, which when auto-activated forms factor XIIa (FXIIa). Here, the production of the recombinant FXIIa protease domain (βFXIIa^His) with yields of ∼1–2 mg per litre of insect-cell culture is reported. A second construct utilized an N-terminal maltose-binding protein (MBP) fusion (MBP-βFXIIa^His). Crystal structures were determined of MBP-βFXIIa^Hisin complex with the inhibitor D-Phe-Pro-Arg chloromethyl ketone (PPACK) and of βFXIIa^Hisin isolation. The βFXIIa^Hisstructure revealed that the S2 and S1 pockets were occupied by Thr and Arg residues, respectively, from an adjacent molecule in the crystal. The Thr-Arg sequence mimics the P2–P1 FXIIa cleavage-site residues present in the natural substrates prekallikrein and FXII, and Pro-Arg (from PPACK) mimics the factor XI cleavage site. A comparison of the βFXIIa^Hisstructure with the available crystal structure of the zymogen-like FXII protease revealed large conformational changes centred around the S1 pocket and an alternate conformation for the 99-loop, Tyr99 and the S2 pocket. Further comparison with activated protease structures of factors IXa and Xa, which also have the Tyr99 residue, reveals that a more open form of the S2 pocket only occurs in the presence of a substrate mimetic. The FXIIa inhibitors EcTI and infestin-4 have Pro-Arg and Phe-Arg P2–P1 sequences, respectively, and the interactions that these inhibitors make with βFXIIa are also described. These structural studies of βFXIIa provide insight into substrate and inhibitor recognition and establish a scaffold for the structure-guided drug design of novel antithrombotic and anti-inflammatory agents.