This study examined whether the forkhead transcription factors of O group 1 (FoxO1) might be involved in telomere biology during calorie restriction (CR). We used FoxO1-knockout heterozygous mice (FoxO1(+/-)) and wild-type mice (WT) as a control. Both WT and FoxO1(+/-) were subjected to ad libitum (AL) feeding or 30 % CR compared to AL for 20 weeks from 15 weeks of age. The heart-to-body weight ratio, blood glucose, and serum lipid profiles were not different among all groups of mice at the end of the study. Telomere size was significantly lower in the FoxO1(+/-)-AL than the WT-AL, and telomere attrition was not observed in either WT-CR or FoxO1(+/-)-CR. Telomerase activity was elevated in the heart and liver of WT-CR, but not in those of FoxO1(+/-)-CR. The phosphorylation of Akt was inhibited and Sirt 1 was activated in heart tissues of WT-CR and FoxO1(+/-)-CR. However, the ratio of conjugated to cytosolic light chain 3 increased and the level of p62 decreased in WT-CR, but not in FoxO1(+/-)-CR. A marker of oxidative DNA damage, 8-OhdG, was significantly lower in WT-CR only. The level of MnSOD and eNOS increased, and the level of cleaved caspase-3 decreased in WT-CR, but not FoxO1(+/-)-CR. Echocardiography showed that the left ventricular end-diastolic and systolic dimensions were significantly lower in WT-CR or FoxO1(+/-)-CR than WT-AL or FoxO1(+/-)-AL, respectively. The present studies suggest that FoxO1 plays beneficial roles by inducing genes involved in telomerase activity, as well as anti-oxidant, autophagic, and anti-apoptotic genes under conditions of CR, and suggest that FoxO1 signaling may be an important mediator of metabolic equilibrium during CR.