Itraconazole inhibited MCF-7 and SKBR-3 cells proliferation via induction of cell death and G0/G1 cell cycle arrest. Itraconazole treatment promoted apoptosis and autophagic cell death, which was regulated by Hedgehog pathway inhibition. Hedgehog pathway inhibitor GANT61 or SHH siRNA synergistically enhanced anti-proliferation effect induced by itraconazole. Anti-breast cancer activity of itraconazole or combined with GANT61 was confirmed in a human xenograft nude mouse model.