An amphiphilic peptide dendrimer conjugated with gemcitabine (GEM), PEGylated dendron-Gly-Phe-Leu-Gly-GEM (PEGylated dendron-GFLG-GEM), is developed as a nano-prodrug for breast cancer therapy. The self-assembled behavior is observed under a transmission electron microscopy and dynamic light scattering. The negatively charged surface and hydrodynamic size of the amphiphilic nanosized prodrug supported that the prodrug can maintain the stability of GEM during circulation and accumulate in the tumor tissue. Drug release assays are conducted to monitor the release of GEM from this nanodrug delivery system in response to the tumor microenvironment, and these assays confirm that GEM released from the nanocarrier is identical to free GEM. The GEM prodrug can prevent proliferation of tumor cells. The therapeutic effect against breast cancer is systematically investigated using an in vivo animal model. Immunohistochemical results are aligned with the significantly enhanced anticancer efficacy of GEM released from the prodrug. This self-assembled amphiphilic drug delivery nanocarrier may broaden the application for GEM and other anticancer agents for breast cancer chemotherapy.