NDR/LATS kinase family is highly conserved from yeast to human. It remains unknown whether the members of this family function in innate immune responses. Here we demonstrate that Stk38 negatively regulates TLR9-mediated immune responses in macrophages. Stk38 constitutively associates with ubiquitin E3 ligase Smurf1, and facilitates Smurf1-mediated MEKK2 ubiquitination and degradation. MEKK2 is required for CpG-induced ERK1/2 activation, TNF-α and IL-6 production but not required for LPS-induced TNF-α and IL-6 production. Accordingly, Stk38 deficiency increases CpG-induced ERK1/2 activation, TNF-α and IL-6 production without significantly affecting LPS-induced TNF-α and IL-6 production. Stk38-deficient mice produce more TNF-α and IL-6, and display increased lethality than control wild-type mice upon E. coli infection. Stk38-deficient mice are also more susceptible to CLP-induced sepsis than control mice. Thus, Stk38 is important in limiting inflammatory cytokine production and necessary for protecting host from inflammatory injury during infection, possibly by negatively regulating TLR9 signalling.