We constructed a recombinant Mycobacterium bovis bacillus Calmette-Guérin (BCG-ΔUT) that lacks urease, providing acidic intraphagosomal conditions to drive an effective human immune T-cell response. BCG-ΔUT-infected macrophages stimulated autologous CD4+ T cells more efficiently than parent BCG-infected macrophages. For further T-cell activation, BCG-ΔUT-infected macrophages required pretreatment with exogenous recombinant granulocyte-macrophage colony-stimulating factor or costimulation with either CD40 ligand or interferon-γ. By contrast, BCG-ΔUT-infected dendritic cells induced significant activation of naïve CD4+ T cells without costimulating signals. C57BL/6 mice intradermally inoculated with BCG-ΔUT more efficiently produced memory T cells that responded to recall antigen. Therefore, the depletion of urease from BCG is useful for the activation of T cells.