A patient with severe haemophilia B with a glycine-to-valine missense mutation at residue 190 (c25, chymotrypsin numbering) in factor IX (FIX; FIX-G190V or FIX-FuChou) had <1% of normal FIX clotting activity and 36% of normal FIX antigen levels (cross-reacting material- reduced, CRMr). Residue 190 in the C-terminal protease domain of human FIX is highly conserved in mammalian species and the serine protease family, suggesting that it has an indispensable role in protein function. To explore the pathological mechanism by which this mutation contributes to dysfunction of the FIX molecule, we functionally characterised FIX-G190V in vitro and in vivo. Liver-specific FIX-G190V gene expression following hydrodynamic plasmid delivery into haemophilia B mice revealed a 5.7-fold reduction in specific clotting activity compared with FIX-WT (wild type) and a two-fold decrease in plasma FIX-G190V concentration. Pulse-chase analysis demonstrated that FIX-G190V was secreted at a significantly slower rate than was FIX-WT. Purified FIX-G190V and FIX-WT displayed normal calcium-dependent conformational changes as shown by intrinsic fluorescence quenching. The in vivo half-lives of FIX-G190V and FIX-WT were indistinguishable. FIX-G190V was, however, more readily degraded than FIX-WT, especially after being activated by the active form of FXI. The vulnerable sites were mapped to the peptide bonds at Arg¹¹⁶-Leu¹¹⁷, Lys²⁶⁵-Tyr²⁶⁶, Arg³²⁷-Val³²⁸, and Arg³³⁸-Ser³³⁹, which are in the exposed loops of the FIX molecule. Also, failure of FXIa-activated FIX-G190V to bind p-aminobenzamidine indicated an abnormal conformation of the active-site pocket. Thus, the mutation at residue 190 of FIX may result in protein misfolding that affects secretion, clotting function, and hydrolysis.