Tat peptides are useful carriers for delivering biologic molecules into the cell for both functional analysis of intracellular disease-related proteins and treatment of refractory diseases. Most internalized Tat-fused cargos (Tat-cargos) are trapped within the endosome, however, which limits the biologic function of the cargo. In this study, we demonstrated that Tat-fused HA2 peptide (HA2Tat), an endosome disrupted peptide, enhanced the endosome-escape efficiency of Tat-cargos. In cells treated with a mixture of fluorescein isothiocyanate-labeled Tat and HA2Tat, widespread fluorescence was observed throughout the cytosol. In addition, this HA2Tat-mediated cytosolic delivery technique led to enhanced cytotoxicity of Tat-fused anti-cancer peptides, specifically shepherdin. Thus, we improved the function of the delivered molecules by co-treating with HA2Tat and propose that this is a useful method for the delivery of therapeutic macromolecules into the cytosol.