Degradation of the extracellular matrices in the human body is controlled by matrix metalloproteinases (MMPs)， a family of more than 20 homologous enzymes. Imbalance in MMP activity can result in many diseases， such as arthritis， cardiovascular diseases， neurological disorders， fibrosis， and cancers. Thus， MMPs present attractive targets for drug design and have been a focus for inhibitor design for as long as 3 decades. Yet， to date， all MMP inhibitors have failed in clinical trials because of their broad activity against numerous MMP family members and the serious side effects of the proposed treatment. In this study， we integrated a computational method and a yeast surface display technique to obtain highly specific inhibitors of MMP-14 by modifying the natural non-specific broad MMP inhibitor protein N-TIMP2 to interact optimally with MMP-14. We identified an N-TIMP2 mutant， with five mutations in its interface， that has an MMP-14 inhibition constant ( ) of 0.9 pm， the strongest MMP-14 inhibitor reported so far. Compared with wild-type N-TIMP2， this variant displays ～900-fold improved affinity toward MMP-14 and up to 16，000-fold greater specificity toward MMP-14 relative to other MMPs. In an and cell-based model of MMP-dependent breast cancer cellular invasiveness， this N-TIMP2 mutant acted as a functional inhibitor. Thus， our study demonstrates the enormous potential of a combined computational/directed evolution approach to protein engineering. Furthermore， it offers fundamental clues into the molecular basis of MMP regulation by N-TIMP2 and identifies a promising MMP-14 inhibitor as a starting point for the development of protein-based anticancer therapeutics.