Hepatocyte growth factor activator inhibitor type 1 (HAI-1), which is encoded by the SPINT1 gene, is a membrane-associated serine proteinase inhibitor abundantly expressed in epithelial tissues. We had previously demonstrated that HAI-1 is critical for placental development, epidermal keratinization, and maintenance of keratinocyte morphology by regulating cognate proteases, matriptase and prostasin. After performing ultrastructural analysis of Spint1-deleted skin tissues, our results showed that Spint1-deleted epidermis exhibited partially disrupted epidermal basement-membrane structures. Matrix metalloproteinases-9 (MMP-9) expression levels were upregulated in Spint1-deleted primary cultured keratinocytes and SPINT1 knockout (KO) HaCaT cells. Furthermore, gelatin zymography of the conditioned medium showed increased MMP activities in keratinocytes with reduced HAI-1 expression. Treating SPINT1 KO HaCaT cells with dehydroxymethylepoxyquinomicin (DHMEQ), a small molecule inhibitor of NF-κB, abrogated the upregulation of MMP9 and the gelatinolytic activity associated with MMP-9. These results suggest that HAI-1 may play a critical role in epidermal basement membrane integrity by regulating NF-κB activation-induced upregulation of MMP-9.