Background and Purpose One characteristic of tumor-infiltrating CD4+Foxp3+ regulatory T cells (Tregs) is high expression of tumor necrosis factor receptor II (TNFR2), a receptor that mediates the decisive effect of TNF in the activation and expansion of Tregs. There is increasing evidence that inhibition of TNFR2 can enhance anti-tumor immune responses. Therefore, we screened Chinese herbal extracts for their capacity to blockade of TNF-TNFR2 interaction. Experimental Approach The inhibitory effect of Chinese herbal extracts and their containing compounds on the biological consequence of TNF-TNFR2 interaction was initially examined with WEHI-13VAR cells and then verified by the primary TNFR2+ Tregs. The resultant small molecule TNFR2 inhibitor on the expansion of Tregs in tumor environment was investigated in mouse CT26 colon model. The effect of such compound on the efficacy of CpG oligodeoxynucleotide in the treatment of tumor was further evaluated with same model. Key Results The results showed that one Chinese herb extract was able to inhibit TNF/TNFR2-induced death of WEHI-13VAR cells and TNF-induced proliferation of TNFR2+ Tregs. Our subsequent study found that flavonoid compound scutellarin was responsible for the activity. Importantly, scutellarin treatment markedly enhanced the efficacy of tumor immunotherapy with CpG oligodeoxynucleotide in mouse CT26 colon cancer model. This effect of scutellarin was associated with the reduction of tumor-infiltrating TNFR2-expressing Tregs, and increased tumor infiltration of IFN-–producing CD8+ T cells. Conclusion and Implications Scutellarin has activities in the inhibition of Tregs expansion induced by TNF-TNFR2 interaction. Our result also suggests that scutellarin or its analogues may be useful as an adjuvant to enhance anti-tumor effect of immunotherapy.