Wip1 is a serine/threonine phosphatase encoded by the ppm1d gene and plays important roles in cell differentiation/proliferation, stress response, inflammation as well as tumorigenesis. In this study, we investigated the function of Wip1 in LPS-induced TLR4 signaling and interferon response. Western blotting analysis indicated that LPS-induced phosphorylation of IRF3 was diminished in Wip1-deficient mouse embryonic fibroblasts (MEFs). Moreover, Wip1-deficient MEFs expressed less IFN-β and interferon-stimulated genes (ISGs) Mx1, Mx2 and Rasd2, compared to the wild-type MEFs. Next, we constructed viral vectors expressing wild-type or phosphatase-dead mutant of Wip1 into Wip1-deficient MEFs, and tested their effects on LPS-induced IFN response. Real-time PCR assay showed that both wild-type and phosphatase-dead mutant of Wip1 were able to restore LPS-induced IFN response in Wip1-deficient MEFs, suggesting that Wip1 regulates IFN response independent of its phosphatase activity. Together, these results suggest a novel role for Wip1 in the regulation of IRF3 activation and LPS-induced IFN response.