Autocrine or paracrine signaling by the type I interferon IFNβ is essential for many of 24 the responses of macrophages to pathogen associated molecular patterns. This feedback loop contributes to pathological responses to infectious agents, and is therefore tightly regulated. We demonstrate here that macrophage expression of IFNβ is negatively regulated by mitogen- and stress-activated kinases (MSK) 1 and 2. Lipopolysaccharide- (LPS-) induced expression of IFNβ was elevated in both MSK1/2 knockout mice and macrophages. Although MSK1 and 2 promote the expression of the anti-inflammatory cytokine interleukin 10, this did not strongly contribute to the ability of MSKs to regulate IFNβ expression. Instead, MSK1 and 2 inhibit IFNβ expression via the induction of dual specificity phosphatase 1 (DUSP1), which dephosphorylates and inactivates the mitogen-activated protein kinases p38 and JNK. Prolonged LPS-induced activation of p38 and JNK, phosphorylation of downstream transcription factors, and over-expression of IFNβ mRNA and protein were similar in MSK1/2 and DUSP1 knockout macrophages. Two distinct mechanisms were implicated in the overexpression of IFNβ: first, JNK mediated activation of c-jun, which binds to the IFNβ promoter and second, p38-mediated inactivation of the mRNA-destabilizing factor tristetraprolin, which we show is able to target the IFNβ mRNA.