Boletus bicolor is a kind of wild edible boletus, which is widely distributed in China. We have isolated the protein D1 from B. bicolor, and found that protein D1 has a significant inhibitory effect on the growth and proliferation of lung cancer A549 cells both in vitro and in vivo. In this paper, we found that miR-34a plays an important role in inhibiting the proliferation of A549 cells. miR-34a is the functional target of protein D1 against A549 cells. Protein D1 induces the positive feedback regulation of miR-34a and p53, which further regulates downstream Bcl-2 and CDK6, and inhibits the proliferation of A549 cells.