We carried out a proteomic analysis of THP-1-derived macrophages with and without Brucella abortus A19 (B. abortus A19) infection in order to study the cellular responses to B. abortus A19. The proteins were analyzed at different time points after infection with 2DE followed by MALDI-TOF/TOF identification. Comparative analysis of multiple 2DE gels revealed that the majority of changes in protein abundance appeared between 48 and 96 h after infection. MS identified 44 altered proteins, including 20 proteins increased in abundance and 24 proteins decreased in abundance, which were found to be involved in cytoskeleton, signal transduction, energy metabolism, host macromolecular biosynthesis, and stress response. Moreover, 22 genes corresponding to the altered proteins were quantified by real-time RT-PCR to examine the transcriptional profiles between infected and uninfected THP-1-derived macrophages. Finally, we mapped the altered pathways and networks using ingenuity pathway analysis, which suggested that the altered protein species were heavily favored germ cell-Sertoli cell junction signaling as the primary pathway. Furthermore, mechanisms of viral exit from host cell and macrophage stimulating protein-recepteur d'origine nantais signaling appeared to be major pathways modulated in infected cells. This study effectively provides useful dynamic protein-related information concerning B. abortus infection in macrophages.