Abstract While miRNAs are increasingly linked to various immune responses, whether they can be targeted for regulating in vivo inflammatory processes such as endotoxin-induced Gram-negative sepsis is not known. Production of cytokines by the dendritic cells (DCs) plays a critical role in response to endotoxin, lipopolysaccharide (LPS). We profiled the miRNA and mRNA of CD11c+ DCs in an unbiased manner and found that at baseline, miR-142-3p was among the most highly expressed endogenous miRs while IL-6 was among the most highly expressed mRNA after LPS stimulation. Multiple computational algorithms predicted the IL-6 3′ untranslated region (UTR) to be a target of miR-142-3p. Studies using luciferase reporters carrying wild-type (WT) and mutant IL-6 3′UTR confirmed IL-6 as a target for miR-142-3p. In vitro knockdown and overexpression studies demonstrated a critical and specific role for miR142-3p in regulating IL-6 production by the DCs after LPS stimulation. Importantly, treatment of only WT but not the IL-6–deficient (IL-6−/−) mice with locked nucleic acid (LNA)–modified phosphorothioate oligonucleotide complementary to miR 142-3p reduced endotoxin-induced mortality. These results demonstrate a critical role for miR-142-3p in regulating DC responses to LPS and provide proof of concept for targeting miRs as a novel strategy for treatment of endotoxin-induced mortality.