The　urokinase　plasminogen　activator　(uPA)　plays　a　critical　role　in　tumor　cell　invasion　and　migration　and　is　a　promising　antimetastasis　target.　6-Substituted　analogues　of　5-N,N-(hexamethylene)amiloride　(HMA)　are　potent　and　selective　uPA　inhibitors　that　lack　the　diuretic　and　antikaliuretic　properties　of　the　parent　drug　amiloride.　However,　the　compounds　display　pronounced　selectivity　for　human　over　mouse　uPA,　thus　confounding　interpretation　of　data　from　human　xenograft　mouse　models　of　cancer.　Here,　computational　and　experimental　findings　reveal　that　residue　99　is　a　key　contributor　to　the　observed　species　selectivity,　whereby　enthalpically　unfavorable　expulsion　of　a　water　molecule　by　the　5-N,N-hexamethylene　ring　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　will　guide　further　optimization　of　dual　potent　human/mouse　uPA　inhibitors　from　the　amiloride　class　as　antimetastasis　drugs.