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The serine protease urokinase plasminogen activator (uPA) plays a critical role in tumour cell invasion and migration and is a promising anti-metastasis drug target. 6-Substituted analogues of 5-N,N-(hexamethylene)amiloride (HMA) are potent uPA inhibitors that show anti-metastatic effects in vivo and high selectivity over closely related trypsin-like serine proteases, while lacking the diuretic and anti-kaliuretic properties of the parent drug amiloride. However, the compounds as a class display pronounced selectivity for human over mouse uPA, thus confounding interpretation of data from human xenografted mouse models of cancer. To understand the molecular basis of this selectivity, we performed molecular dynamics simulations and alchemical free energy perturbation calculations using human and mouse uPA and their complexes with amiloride, HMA and 6-substituted HMA analogues and compared the results to enzyme inhibitory potencies. X-ray structures of selected compounds bound to partially murinised human uPA (H99Y) were also obtained. Collectively, the computational and experimental findings revealed that residue 99 is a key contributor to human/mouse uPA species selectivity, whereby enthalpically unfavourable steric expulsion of a water molecule by the 5-N,N-hexamethylene ring of HMA and analogues occurs when residue 99 is Tyr (as in mouse uPA). Analogue 7 lacking the 5-N,N-hexamethylene ring maintained similar water networks when bound to human and mouse uPA and displayed reduced selectivity, thus supporting this conclusion. The study highlights the crucial role water molecules can play in protein ligand binding and will serve to guide further optimisation of dual-potent human/mouse uPA inhibitors from the amiloride class as anti-metastasis drugs.