Bacterial cell wall component-induced tolerance represents an important protective mechanism during microbial infection. Tolerance induced by the TLR2 agonist bacterial lipoprotein (BLP) has been shown to attenuate the inflammatory response, and simultaneously to augment antimicrobial function, thereby conferring its protection against microbial sepsis. However, the underlying mechanism by which BLP tolerance augments bactericidal activity has not been fully elucidated. Here, we reported that the induction of BLP tolerance in murine macrophages upregulated the expression of Rab20, a membrane trafficking regulator, at both the mRNA and protein levels upon bacterial infection. The knockdown of Rab20 with Rab20 specific siRNA (siRab20) did not affect the phagocytosis of Escherichia coli (E. coli), but substantially impaired the intracellular killing of the ingested E. coli in BLP-tolerized macrophages. Furthermore, Rab20 was associated with GFP-E. coli containing phagosomes, and BLP tolerization resulted in the enhanced maturation of GFP-E. coli-containing phagosomes associated with Rab20 and strong lysosomal acidification. The knockdown of Rab20 substantially diminished lysosome acidification and disturbed the fusion of GFP-E. coli containing phagosomes with lysosomes in BLP-tolerized macrophages. These results demonstrate that Rab20 plays a critical role in BLP tolerization-induced augmentation of bactericidal activity via promoting phagosome maturation and the fusion of bacteria containing phagosomes with lysosomes.