CD4⁺ and CD8⁺ T-cell exhaustion developed in chronic viral infection has been important issue because exhausted antigen-specific CD4⁺ and CD8⁺ T cells showed impaired ability to eradicate persistently infected virus and produce effector cytokines such as IFN-γ and TNF-α. Hence, strategies that either restore endogenous exhausted T-cell responses or provide functional CD4⁺ and CD8⁺ T cells need to be urgently developed for therapeutics of human chronic infection. In spite of promising development using antibody and cell immunotherapy, there were no attempts to restore exhausted CD4⁺ and CD8⁺ T cells using treatment of small bioactive molecules, especially derived from natural resources. Here, using mouse model of chronic infection with lymphocytic choriomeningitis virus (LCMV), we found for the first time that genkwadaphnin isolated from dried flower buds of Daphne genkwa (Thymelaeceae) significantly restored exhausted CD4⁺ and CD8⁺ T cells, as corroborated by evidences that genkwadaphnin treatment enhanced functional LCMV-specific CD4⁺ and CD8⁺ T cells in both quantitative and qualitative. Repeated administration of lower genkwadaphnin doses also induced recovery of exhausted LCMV-specific CD4⁺ and CD8⁺ T cells. Furthermore, pretreatment of genkwadaphnin prior to LCMV infection prevent the generation of exhausted LCMV-specific CD8⁺ T cells. Such restoration of exhausted LCMV-specific CD4⁺ and CD8⁺ T cells by genkwadaphnin was closely associated with reduction of viral burden in sera as well as tissues. More intriguingly, genkwadaphnin treatment induced down-regulation of negative regulatory molecules such as PD-1 and Tim-3 in exhausted LCMV-specific CD4⁺ and CD8⁺ T cells with more apparent down-regulation of Tim-3, which suggesting that Tim-3 molecule is main target to restore exhausted T-cell responses with genkwadaphnin. Collectively, these results provide new valuable insight into the use of genkwadaphnin isolated form flos buds of Daphne genkwa to develop therapeutic strategy of chronic human diseases, such as hepatitis B and C virus (HBV and HCV), human immunodeficiency virus (HIV) as well as cancers.