Advanced glycation end products (AGEs) are elevated in aged and diabetic individuals and are associated with pathologic changes associated with both. Previously we demonstrated that the AGE, CML-collagen, induced fibroblast apoptosis through the cytoplasmic and mitochondrial pathways and the global induction of pro-apoptotic genes. Here we investigated upstream mechanisms of CML-collagen induced apoptosis. CML-collagen induced activation of the proapoptotic transcription factor FOXO1 compared to unmodified collagen. When FOXO1 was silenced CML-collagen stimulated apoptosis was reduced by approximately 75% compared to fibroblasts incubated with non-silencing siRNA demonstrating the functional significance of FOXO1 activation (P<0.05). CML-collagen but not control collagen also induced a 3.3 fold increase in p38 and a 5.6 fold increase in JNK(1/2) activity (P<0.05). By use of specific inhibitors activation of p38 and JNK were shown to play an important role in CML-collagen induced activation of FOXO1 and caspase-3. Moreover, inhibition of p38 and JNK reduced CML-collagen stimulated apoptosis by 48% and 57% percent respectively and by 89% when used together (P<0.05). In contrast, inhibition of the PI3 kinase/Akt pathway enhanced FOXO1 activation. p38 and JNK stimulation by CML-collagen was almost entirely blocked when formation of reactive oxygen species was inhibited and partially reduced by nitric oxide (NO) and ceramide inhibitors. These inhibitors also reduced apoptosis to a similar extent. Taken together these data support a model in which AGE induced apoptosis involves activation of the ROS, NO, ceramide and lead to p38 and JNK MAP kinase activation, which in turn induces FOXO1 and caspase-3.