A novel serine protease inhibitor gene was isolated from Hevea brasiliensis leaves, a RRIT251 cultivar and designated RRIT251 H. brasiliensis protease inhibitor (251Hbpi). Reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) were used to isolate 251Hbpi. A full-length cDNA of 251Hbpi encoded a 70 amino acid protein. 251HbPI is a member of the potato inhibitor I (PI-I) family of serine protease inhibitors. The amino acid residues at the active site of 251HbPI were predicted as Met 46-Glu 47. Multiple alignments of the homologous PI-I family revealed one motif WPELVG of 251HbPI conserved across the family. 251Hbpi was cloned into expression vector pFLAG-ATS and expressed in Escherichia coli strain BL21. Molecular weight of the recombinant 251HbPI (r251HbPI) was approximately 11 kDa. Protease inhibition analysis revealed that r251HbPI inhibited the activity of chymotrypsin and subtilisin A but did not trypsin protease. Moreover, purified r251HbPI protein inhibited Trichophyton rubrum with a minimum inhibitory concentration of 0.7 mg/ml and a minimum fungicidal concentration of 1.4 mg/ml. The specific T. rubrum protease targets of r251HbPI were analyzed by co-immunoprecipitation. r251HbPI interacted with approximate 27 and 61 kDa T. rubrum proteins, suggesting a role in the inhibition of T. rubrum growth. These results suggest that 251HbPI could be a candidate for the development of a novel drug to treat T. rubrum infection.