MAP kinases are downstream targets of immune receptor signaling, having essential roles in both innate and adaptive immunity. The activities of MAP kinase signaling pathways in immune responses are tightly regulated by various mechanisms to ensure proper biological outcomes. One protein family known as MAP kinase phosphatases (MKPs) or dual specificity phosphatases (DUSPs) plays essential roles in negative regulation of MAP kinase activation. There are 10 typical and 16 atypical MKPs have been identified. DUSP12 is one of the atypical MKPs whose substrate and function are unknown. To examine the regulation of MAP kinase activation in immune responses by DUSP12, we cloned mouse DUSP12 full length cDNA. We found that when overexpressed in 293T cells, DUSP12 interacts with ERK, JNK and p38. However, it inhibits the activation of p38 and, to a less extent, JNK, but not ERK in macrophages in response to TLR activation. Overexpression of DUSP12 inhibits the expression and production of inflammatory cytokines such as TNFa and MCP-1 in response to various TLR activation and intracellular pathogen infection. The regulation of MAP kinase activation by DUSP12 and the function of this protein in macrophage activation and function were further investigated. Our results demonstrated that DUSP12 is a bona fide MAP kinase phosphatase, plays important roles in anti-microbial infection in macrophages.