Tetrandrine (TET) is an anti-inflammatory compound isolated from Chinese herb Stephania tetrandra S. Moore. It was reported recently that the differentiation of Th17 cells was inhibited, while the generation of induced Treg cells (iTregs) was promoted, by TET treatment. We therefore carefully examined the effect of TET on the differentiation of four major subsets of T helper cells. The results showed that in vitro treatment with TET potently inhibited the differentiation of Th1, Th2 and Th17 cells. Administration of LPS resulted in a mixed Th1, Th2 and Th17 responses in normal mice, and such effect of LPS was inhibited by in vivo TET treatment as well. In contrast, TET did not promote or inhibit the in vitro generation of iTregs from naïve CD4+CD25-Foxp3/gfp- T cells. Furthermore, spontaneous and rapamycin-induced conversion of naïve CD4+CD25-Foxp3/gfp- T cells into Foxp3-expressing iTregs in congenic mice was not affected by TET treatment. Thus, TET had the capacity to inhibit the differentiation of proinflammatory Th1, Th2 and Th17 cells, while sparing the generation of Tregs. As a Treg-friendly and broad spectrum anti-inflammatory agent, the molecular mechanism and the therapeutic potential of TET in various human inflammatory diseases should be further studied.