TonEBP is an osmosensitive transcription factor with essential functions in the urinary concentrating process and osmoadaptation of renal medullary cells. The present study addressed the effect of focal adhesion kinase (FAK), a mechanosensitive non-receptor tyrosine kinase abundantly expressed along medullary nephron segments, on TonEBP transcriptional activity and target gene expression. In HEK293 cells exposed to osmotic stress, the small molecule FAK inhibitor PF-228 dose-dependently blunted TonE-driven reporter activity, aldose reductase (AR) promoter activity, and expression of TonEBP target genes AR and HSP70, respectively. Accordingly, these effects could be reproduced by knockdown of FAK in HEK293 cells by siRNA transfection. Inhibition of FAK had no effect on nuclear redistribution of TonEBP under hypertonic conditions. Furthermore, TonEBP transactivation activity, as assessed by the Gal4 binary assay, was not influenced by FAK inhibition. In contrast, FAK inhibition was associated with strongly reduced TonEBP mRNA and protein levels, which resulted from FAK-dependent stimulation of TonEBP gene transcription and not from reduced TonEBP mRNA or protein half life. In conclusion, the present study identifies FAK as a major regulator of TonEBP activity by virtue of increasing its expression, thereby contributing essentially to TonEBP transcriptional activity.