20(R)-ginsenoside Rg3 (20(R)-Rg3) has shown multiple pharmacological activities and been considered as one of the most promising approaches for fatigue treatment. However, 20(R)-Rg3 has a low bioavailability after oral administration in human due to the first-pass effect. Recently, nasal route has gained increasing interest as it can avoid first-pass effect for its lower enzymatic activity compared with the gastrointestinal tract and liver. In order to provide an animal experimental evidence of 20(R)-Rg3 intranasal administrated preparation, the anti-fatigue effect of 20(R)-Rg3 after intranasal administration was investigated. Two weeks after 20(R)-ginsenoside Rg3 was administrated intranasally to mice at three different doses, the anti-fatigue effect of 20(R)-Rg3 was evaluated by the weight-loaded swimming test and biochemical parameters related to fatigue, such as serum urea nitrogen (SUN), lactic dehydrogenase (LDH), superoxide dismutase (SOD), malondialdehyde (MDA), blood lactic acid (LA) and hepatic glycogen. The results showed that compared with the negative control group, the intermediate-dose and the high-dose groups significantly prolonged the weight-loaded swimming time (p<0.05; p<0.01), and also increased the hepatic glycogen levels (p<0.05); SUN levels were decreased considerably in three 20(R)-Rg3-treated groups (p<0.01). In addition, the low-dose group obviously decreased the content of blood LA (p<0.05). However, the levels of LDH, SOD and MDA did not show a significant change. Our results predicted a benefit of 20(R)-Rg3 as an anti-fatigue treatment by intranasal administration. The mechanism was related to the increase of the storage of hepatic glycogen, and the decrease of the accumulation of metabolite such as lactic acid and serum urea nitrogen.