Background/Purpose: Aging-associated changes in articular chondrocytes represent a major risk factor for cartilage degradation and osteoarthritis (OA). The major signaling pathway that regulates cellular aging and stress resistance is the Insulin/IGF-1 signaling pathway. In this pathway, Forkhead-box class O (FoxO) transcription factors play central roles in protein quality control, autophagy and cellular defenses against oxidative stress. The objective of this study was to analyze expression and activation of FoxO transcription factors in normal, aging and OA cartilage. Methods: Human knee joints from individuals ages 23-90 were obtained at autopsy. The entire femoral condyles of knee joints were harvested from 6 young normal donors (mean ± SD = 36.0 ± 9.6 years old, OA grade I). Aged knee joints were also obtained from 4 donors (mean ± SD = 72.5 ± 3.6 years) with no history of arthritis and normal or minimally fibrillated cartilage (OA grade I-II). OA human joints were obtained from 4 donors (mean ± SD = 81.5 ± 10.3 years, OA grade III-IV). Knee joints were also obtained from mice at the age of 4, 12, and 24 months and following surgically induced OA. Expression of FoxO proteins was analyzed by immunohistochemistry and western blotting. Regulation of FoxO protein expression and activation was analyzed in cultured chondrocytes stimulated with cytokines, growth factors and oxidants. Results: Human cartilage expressed predominantly FoxO1 and FoxO3 but not FoxO4 protein as detected by western blotting. Immunohistochemistry showed that normal human articular cartilage expressed FoxO1 and FoxO3 proteins more strongly in chondrocytes in the superficial and mid zone as compared to the deep zone. FoxO showed mainly nuclear localization in young normal cartilage. Areas of cartilage exposed to minimal versus maximal weightbearing showed no significant difference in young normal cartilage except for the deep zone where FoxO3 was more strongly expressed. During human joint aging, expression of FoxO1 and FoxO3 was markedly reduced in the superficial zone of cartilage regions exposed to maximal weightbearing. In OA-affected cartilage, chondrocyte clusters showed strong FoxO phosphorylation and cytoplasmic localization. Similar patterns of FoxO expression in normal joints and changes in aging and OA joints were observed in mouse models. In cultured chondrocytes, the longer-term (2-5 day) treatment with proinflammatory cytokines IL-1β and TNF-α suppressed FoxO1 protein, while TGF-β and PDGF increased FoxO1 and FoxO3 expression, respectively. FoxO1 and FoxO3 phosphorylation was increased significantly by short-term (60 minutes) exposure to IL-1β, PDGF, b-FGF, IGF-1, and the oxidant t-BHP. Conclusions: Normal articular cartilage has a tissue specific signature of FoxOs expression and activation. Aging and OA in humans and mice is associated with profound changes in FoxO expression and activation. These results suggest that changes in FoxO expression and activation may be involved in cartilage aging and OA, and provide a foundation upon which the function of FoxOs in cartilage homeostasis, aging and OA can be examined.