To examine the active site of recombinant buckwheat trypsin inhibitor (rBTI), two mutants (R45A-aBTI, R45F-fBTI) were generated through site-directed mutagenesis. Activity analysis found that the aBTI and fBTI had lost trypsin inhibitory activity. However, aBTI and fBTI showed new inhibitory activities against elastase and chymotrypsin, respectively. This result suggested that Arg⁴⁵ is the active site of rBTI. These inhibitors showed remarkable stability to heat and pH. The possible effects of aBTI and fBTI on the proliferation of human HL-60 and EC9706 cell lines were investigated by MTT assays. It indicated that aBTI and fBTI could specifically inhibit the growth of HL-60 and EC9706 cells in a dose- and time-dependent manner. The active site of rBTI was determined and two new inhibitors were obtained in this research. Knowledge about the active site is useful for further clarifying the physiological mechanism of rBTI and other protease inhibitors. New inhibitory activities of aBTI and fBTI are potentially useful in the fields of health and medicine.