Vaccination with Mycobacterium bovis bacille Calmette-Guérin (BCG) has variable efficacy in preventing tuberculosis. Both BCG strain and route of administration have been implicated in determining efficacy; however, these variables are not considered in current clinical recommendations for vaccine choice. We evaluated antigen-specific immunity after percutaneous or intradermal administration of Japanese BCG or intradermal administration of Danish BCG. Ten weeks after vaccination of neonates, percutaneous Japanese BCG had induced significantly higher frequencies of BCG-specific interferon-γ-producing CD4⁺ and CD8⁺ T cells in BCG-stimulated whole blood than did intradermal Danish BCG. Similarly, percutaneous vaccination with Japanese BCG resulted in significantly greater secretion of the T helper 1-type cytokines interferon-γ, tumor necrosis factor-α, and interleukin-2; significantly lower secretion of the T helper 2-type cytokine interleukin-4; and greater CD4⁺ and CD8⁺ T cell proliferation. Thus, BCG strain and route of neonatal vaccination confer different levels of immune activation, which may affect the efficacy of the vaccine.