Host plant protease inhibitors offer resistance to proteases from invading pathogens. Trypsin inhibitors (TIs), in particular, serve as protective agents against insect and pathogen attacks. In this study, we designed a pair of degenerate primers based on highly conserved motifs at the N- and C-termini of the TI from tartary buckwheat (Fagopyrum tataricum; Ft) to clone the central portion. Genomic walking was performed to isolate the 5′ and 3′ flanking regions of FtTI. We demonstrated the successful PCR amplification of a 644 bp portion of FtTI. The full-length DNA of FtTI contains a complete open reading frame of 264 bp, encoding 87 amino acids with a mass of approximately 9.5 kDa. The FtTI protein sequence was 49% identical and 67% similar to potato protease inhibitors. Site-directed mutagenesis identified the residues, Asp67 and Arg68, as crucial for the inhibitory activity of the FtTI. Recombinant and mutant FtTI inhibited both the hyphal growth and spore germination of Alternaria solani. The calculated 50% inhibitory concentrations of FtTI ranged from 5–100 μg mL−1 for spore germination and 1–50 μg mL−1 for fungal growth. Thus, recombinant FtTI may function in host resistance against a variety of fungal plant pathogens.