The kallikrein (KLK) family of proteases is involved in many aspects of human health and disease. This family is highly related and many have a trypsin-like specificity for lys/arg at the P1 site. Since KLK4 has been implicated in cancer development and metastasis, determining mechanisms of inactivation are critical to developing selective KLK4 inhibitors. X-ray crystal structures of KLK4 in complex with sunflower trypsin inhibitor-1 (SFTI-1), a semi-rationally designed SFTI-1 derivative and KLK4 with nickel were determined. This analysis provides atomic-level characterization of the KLK4/inhibitor interaction and a molecular rationalization for the potency and selectivity of these inhibitors. In our Ni-KLK4 structure, an alternate conformation of the Asn192-Gly193 peptide bond is seen that predicts reduced catalysis, weakening of the oxyanion hole and release of Ile16. Additionally, an unstructured loop that is part of the inhibitory metal binding site is observed. Using this structural information in combination with molecular dynamics simulations we show that differential motions are seen upon metal binding and are transmitted to the active site, giving a more detailed structural basis for inhibition. Collectively, this work provides insight into both direct and indirect mechanisms of inhibition for KLK4 that are applicable to the serine protease superfamily.