Background: Aging is a multifactorial degenerative process that can be modulated by fasting through activation of the Fork-head transcription factor of the O class 3 (FOXO3), which plays an important role in increasing lifespans. However, the effects of different fasting durations on the expression of FOXO3 in the liver has not yet been reported. Objective: This study analyzed the effects of different fasting durations on the FOXO3 expression and its pathway by measuring sirtuin1 (SIRT1), insulin-like growth factor-1 (IGF-1), and superoxide dismutase (SOD) activity in the liver. Methods: New Zealand white rabbits were used to mimic the effects of fasting on humans. The rabbits were divided into the control, intermittent fasting (IF), and prolonged fasting (PF) groups. Both fasting groups were interspersed with the non-fasting phase for 8 h. This treatment was conducted for 6 days. On Day 7, all the rabbits were sacrificed, and their livers were taken to measure the FOXO3 and SIRT1 mRNA expressions, the IGF-1 protein level, and the SOD activity level. ANOVA, multiple comparison, and Pearson's correlation were performed for statistical analysis. Results: The FOXO3 and SIRT1 mRNA expressions were significantly higher in the IF group than in the control group. The FOXO3 expression was also 2.5 times higher in the IF group than in the PF group. There was a positive correlation between the FOXO3 and SIRT1 mRNA expressions. The IGF-1 protein level was significantly lower in the IF and PF groups than in the control group. The SOD-specific activity level was significantly higher in the IF group than in the control and PF groups. Conclusions: Intermittent fasting significantly increased the FOXO3 and SIRT1 mRNA expressions and the SOD activity level in the livers of the rabbits and significantly decreased the circulating and hepatic IGF-1. Therefore, intermittent fasting may give a protective intervention effect towards aging.