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Mutations of SPINT2, the gene encoding the integral membrane, Kunitz-type serine inhibitor HAI-2, primarily affect the intestine, while sparing many other HAI-2-expressing tissues, causing sodium loss in patients with syndromic congenital sodium diarrhea. The membrane-bound serine protease prostasin was previously identified as a HAI-2 target protease in intestinal tissues but not in the skin. In both tissues, the highly related inhibitor HAI-1 is, however, the default inhibitor for prostasin and the type 2 transmembrane serine protease matriptase. This cell-type selective functional linkage may contribute to the organ-selective damage associated with SPINT 2 mutations. To this end, the impact of HAI-2 deletion on matriptase and prostasin proteolysis was, here, compared using Caco-2 human colorectal adenocarcinoma cells and HaCaT human keratinocytes. Greatly enhanced prostasin proteolytic activity with a prolonged half-life and significant depletion of HAI-1 monomer were observed with HAI-2 loss in Caco-2 cells but not HaCaT cells. The constitutive, high level prostasin zymogen activation observed in Caco-2 cells, but not in HaCaT cells, also contributes to the excessive prostasin proteolytic activity caused by HAI-2 loss. HAI-2 deletion also caused increased matriptase zymogen activation, likely as an indirect result of increased prostasin proteolysis. This increase in activated matriptase, however, only had a negligible role in depletion of HAI-1 monomer. Our study suggests that the constitutive, high level of prostasin zymogen activation and the cell-type selective functional relationship between HAI-2 and prostasin renders Caco-2 cells more susceptible than HaCaT cells to the loss of HAI-2, causing a severe imbalance favoring prostasin proteolysis.