The mechanical regulation of the forkhead box O (FOXO) subclass of transcription factors in the respiratory pump and its implication in aging are completely unknown. We investigated the effects of diaphragm stretch on three FOXO isoforms, FoxO1, FoxO3a and FoxO4 at different ages of normal mice. We tested the hypotheses that 1) FOXO activities are regulated in response to diaphragm stretch and 2) mechanical properties of aging diaphragm are altered leading to altered regulation of FOXO with aging. Our results showed that stretch downregulated FOXO DNA binding activity by a mechanism that required Akt and IkappaB kinase (IKK) activation in young mice but loss of mechanosensitive of these pathways occurred with age. This aberrant regulation of FOXO with aging was associated with altered viscoelasticity, compliance and extensibility of the aged diaphragm. Curiously, the nuclear content of Foxo1 and Foxo3a , the two isoforms associated with muscle atrophy, diminished dramatically with aging in correlation with higher basal activation of Akt and IKK signaling in diaphragms of old mice . In contrast, the stability of Foxo4 in the nucleus became dependent on JNK, which is strongly activated in aged diaphragm. This suggested that Foxo4 was responsible for the FOXO-dependent transcriptional activity in aging diaphragm. Our data supported the hypothesis that aging alters the mechanical properties of the respiratory pump leading to altered mechanical regulation of the stretch-induced signaling pathways controlling FOXO activities. Our study supports a mechanosensitive signaling mechanism that is responsible for the regulation of the FOXO transcription factors by aging.