Our previous investigation demonstrated that ginsenoside Rg3 was active in promotion of the immune response. In this study, two epimers, 20(R)-Rg3 and 20(S)-Rg3, were compared for their adjuvant effects on the immune response against ovalbumin (OVA). BALB/c mice were immunized subcutaneously with 10 μg of OVA alone or with 10 μg of OVA mixed in 20(R)-Rg3 (50 μg) or 20(S)-Rg3 (50 μg) on days 1 and 15. Two weeks after the last immunization, blood was sampled to test IgG and the IgG subclasses as well as IFN-γ and IL-5; splenocytes were prepared to measure proliferative responses to stimulations with Con A, LPS and OVA and mRNA expressions of cytokines and transcription factors by reverse transcription-PCR. Results indicated that both 20(R)-Rg3 and 20(S)-Rg3 exhibited the adjuvant effect on OVA-induced immune responses. 20(R)-Rg3 promoted significantly higher serum-specific IgG and the IgG isotype responses in association with highly up-regulated serum IFN-γ and IL-5 than 20(S)-Rg3. In addition, 20(R)-Rg3 significantly enhanced splenocyte proliferative responses to Con A, LPS and OVA as well as mRNA expression of IFN-γ, IL-12, IL-4 and IL-10 and transcription factors T-bet and GATA-3 by splenocytes when compared with the 20(S)-Rg3. Therefore, ginsenoside Rg3 is stereospecific in stimulation of the immune response, and 20(R)-Rg3 has more potent adjuvant activity than 20(S)-Rg3.