P. gingivalis is a prominent periodontal pathogen that has potent effects on host cells. In this study we challenged gingival epithelial cells with P. gingivalis with the aim of assessing how mRNA levels of key target genes were modulated by P. gingivalis via the transcription factors FOXO1 and FOXO3. Primary mono- and multi-layer cultures of gingival epithelial cells were challenged and barrier function was examined by fluorescent dextran and apoptosis was measured by cytoplasmic histone associated DNA. Gene expression levels were measured by real-time PCR with and without FOXO1 and FOXO3 siRNA compared to scrambled siRNA. P. gingivalis induced a loss of barrier function and stimulated gingival epithelial cell apoptosis in multilayer cultures that was in part gingipain dependent. P. gingivalis stimulated an increase in FOXO1 and FOXO3 mRNA, enhanced mRNA levels of genes associated with differentiated keratinocyte function (keratin-1, -10, -14, and involucrin), increased mRNA levels of apoptotic genes (BID and TRADD), reduced mRNA levels of genes that regulate inflammation (TLR-2 and -4) and reduced those associated with barrier function (integrin beta-1, -3 and -6). The ability of P. gingivalis to modulate these genes was predominantly FOXO1 and FOXO3 dependent. The results indicate that P. gingivalis has pronounced effects on gingival keratinocytes and modulates mRNA levels of genes that affect host response, differentiation, apoptosis and barrier function. Moreover, this modulation is dependent upon the transcription factors FOXO1 or FOXO3. In addition, a new function for FOXO1 was identified, that of suppressing TLR-2 and TLR-4 and maintaining integrin beta -1, beta -3 and beta -6 basal mRNA levels in keratinocytes.