Panaxydol isolated from the lipophilic fractions of Tienchi ginseng (Panax notoginseng) induces growth inhibition and differentiation of rat C6 glioma cells. The underlying molecular mechanisms are not completely understood. In the present study, we identified phosphatidylinositol 3-kinase (PI 3-K) as a necessary enzyme for the differentiation of C6 cells treated with panaxydol. The specific PI 3-K inhibitor wortmannin resulted in attenuated differentiation of C6 cells induced by panaxydol, and was associated with perinuclear localization of glial fibrillary acidic protein expression and a diminished process formation. These data suggest that induction of differentiation in C6 cells by panaxydol could be mediated through a PI 3-K-dependent pathway.