Silent mating type information regulation 2 homolog 1 (SIRT1) plays a critical role in reactive oxygen species-triggered apoptosis in mouse embryonic stem (mES) cells. Here, we investigated a possible role for the PTEN/Akt/JNK pathway in the SIRT1-mediated apoptosis pathway in mES cells. Akt was activated by removal of anti-oxidant 2-mercaptoethanol in SIRT1(-/-) mES cells. Since PTEN is a negative regulator of Akt and its activity can be modulated by acetylation, we investigated if SIRT1 deacetylated PTEN to downregulate Akt to trigger apoptosis in anti-oxidant-free culture conditions. PTEN was hyperacetylated and excluded from the nucleus in SIRT1(-/-) mES cells, consistent with enhanced Akt activity. SIRT1 deficiency enhanced the acetylation/phosphorylation level of FOXO1 and subsequently inhibited the nuclear localization of FOXO1. Cellular acetylation levels were enhanced by DNA-damaging agent, not by removal of anti-oxidant. c-Jun NH2-terminal kinase (JNK) was activated by removal of anti-oxidant in SIRT1-dependent manner. Although p53 acetylation was stronger in SIRT1(-/-) mES cells, DNA-damaging stress activated phosphorylation and enhanced cellular levels of p53 irrespective of SIRT1, whereas removal of anti-oxidant slightly activated p53 only with SIRT1. Expression levels of Bim and Puma were increased in anti-oxidant-free culture conditions in an SIRT1-dependent manner and treatment with JNK inhibitor blocked induction of Bim expression. DNA-damaging agent activated caspase3 regardless of SIRT1. Our data support an important role for SIRT1 in preparing the PTEN/JNK/FOXO1 pathway to respond to cellular reactive oxygen species.