The Gram-negative bacteria Brucella abortus is a major cause of brucellosis in animals and humans. The host innate immune response to B. abortus is mainly associated with phagocytic cells such as dendritic cells, neutrophils, and macrophages. However, as mast cells naturally reside in the main bacterial entry sites they may be involved in bacterial recognition. At present, little is known about the role of mast cells during B. abortus infection. The role of the innate immune receptors TLR2 and TLR4 in activation of mast cells by B. abortus (strain RB51) infection was analyzed in this study. The results showed that B. abortus did not induce mast cell degranulation, but did induce the synthesis of the cytokines IL-1β, IL-6, TNF-α, CCL3, CCL4, and CCL5. Furthermore, B. abortus stimulated key cell signaling molecules involved in mast cell activation such as p38 and NF-κB. Blockade of the receptors TLR2 and TLR4 decreased TNF-α and IL-6 release by mast cells in response to B. abortus. Taken together, our results demonstrate that mast cells are activated by B. abortus and may play a role in inducing an inflammatory response during the initial phase of the infection.