Macrophage is essential for host anti-bacterial defense by directly eliminating invading microbes and inducing a series of immune reactions. Here we identified a Streptococcus pneumoniae protein, PepO, as a TLR2/TLR4 bi-ligand. We found that PepO enhances macrophage unspecific phagocytosis and bactericidal activity, which is related to the induction of autophagy in macrophage, for the inhibition of autophagy significantly decreased the phagocytosis and bactericidal activity of PepO-treated macrophage. We confirmed that these effects of PepO are dependent on interacting with both TLR2 and TLR4. The tlr2 or tlr4 deficiency partially abolished the effect of PepO while tlr2/tlr4 deficiency abolished it completely. In vivo study demonstrated that PepO reduced the bacteria load in WT mice significantly, while the depletion of macrophage or tlr2/tlr4 deficiency abrogated the effect of PepO. Our findings suggested the therapeutic potential of PepO and provided experimental evidence for immunotherapy against infectious disease.