Technical Abstract: Blue cohosh, Caulophyllum thalictriodes is a popular herb, roots and rhizomes of which have been extensively used for women’s health. Alkaloids and saponins are considered to be responsible for its pharmacological effects. In this investigation the methanolic extract of the roots of blue cohosh, alkaloidal fraction and isolated constituents were evaluated for their inhibition of major drug metabolizing cytochrome P450 (CYP450) enzymes. Methanolic extract did not show any effect but the alkaloidal fraction showed a strong inhibition of CYP 2C19, 3A4, 2D6, and 1A2 (>80% inhibition at 100 µg/mL) with IC50 values in the range of 2 - 20 µg/mL. Among the isolated alkaloids, a piperidine type alkaloid (caulophyllumine B) and three lysine derived alkaloids (O-acetlybaptifolin, anagyrine, and lupanine) inhibited these enzymes to various extents (IC50 2.5 - 50 µM). N-methylcytisine weakly inhibited CYP3A4 only (32% inhibition at 100 µM). An equimolar mixture of alkaloids exhibited more pronounced inhibitory effect on all the four enzymes as compared to the isolated alkaloids. Among the saponins, caulosides C and D inhibited CYP3A4 at the highest test concentration of 100 µM (43% and 35% inhibition, respectively). Other enzymes were not affected. This in vitro study indicates that dietary supplements containing blue cohosh may pose a risk of drug-drug interactions if taken with other drugs or herbs, metabolism of which involves CYP450 enzymes.