The cystatin superfamily includes proteins crucial for inhibiting cysteine proteases, enzymes involved in many biological processes. In plants, cystatins regulate seed germination, development, and pathogen defense. In humans, inhibiting legumain-type cysteine proteases offers a promising cancer treatment strategy, as this enzyme's expression often rises during tumor progression. We evaluated a novel rice-derived chimeric legumain inhibitor using in silico and in vitro methods. Computational simulations confirmed the inhibitor's stability and nanomolar affinity for legumain's active site. Post-expression and purification assays determined its kinetics and demonstrated its efficacy in reducing HT29 tumor cell migration and viability. Our findings suggest the chimeric Oryzacystatin I mutant with SNSL motifs is a promising candidate for cancer drug development.