Mast cells are best known as central effector cells in IgE-mediated type I allergic diseases including asthma and hay fever. An increasing amount of evidence, however, has demonstrated that mast cells are sentinel cells playing a critical role in host defense against invading microbes. Mast cells are located immediately beneath the epithelial surfaces exposed to the outer environment, such as genitourinary and gastrointestinal tracts, skin, and airways. This review discusses recent studies on the critical roles of mast cells in host defense against Gram-negative bacterial infection. Mast cells are equipped with multiple receptors detecting the invading Gram-negative bacteria in both direct (opsonin-independent) and indirect (opsonin-dependent) mechanisms. The former includes Toll-like receptors (TLRs), CD48, and nucleotide-binding oligomerization (NOD) proteins, while the latter includes Fcγ receptors (FcγRs) and complement receptors. In addition to the detecting systems, mast cells are also armed with versatile tools to combat and kill Gram-negative bacteria. In response to the recognition of the Gram-negative bacterial infection, mast cells secrete various types of mediators which either regulate host immune system or directly attack the bacteria. Mast cells can also phagocytize and subsequently display the bacterial antigens on their cell surfaces. Moreover, recent findings have revealed the formation of extra-cellular traps by mast cells. Finally this review will especially focus on recent findings on LPS signaling in mast cells, both the functional outcome and the molecular mechanisms.