D-Fraction is protein-bound β-1,6 and β-1,3 glucans (proteoglucan) extracted from the edible and medicinal mushroom Grifola frondosa (Maitake). The antitumoral effect of D-Fraction has long been exclusively attributed to their immunostimulatory capacity. However, in recent years increasing evidence showed that D-Fraction directly affects the viability of canine and human tumor cells, independent of the immune system. Previously, we have reported that D-Fraction modulates the expression of genes associated with cell proliferation, cell death, migration, invasion, and metastasis in MCF7 human breast cancer cells. Therefore, the purpose of the current study is to investigate if this modulation of gene expression by Maitake D-Fraction really modulates tumor progression. In the present work, we demonstrate for the first time that Maitake D-Fraction is able to act directly on mammary tumor cells, modulating different cellular processes involved in the development and progression of cancer. We demonstrate that D-Fraction decreases cell viability, increases cell adhesion, and reduces the migration and invasion of mammary tumor cells, generating a less aggressive cell behavior. In concordance with these results, we also demonstrate that D-Fraction decreases tumor burden and the number of lung metastases in a murine model of breast cancer.