BACKGROUND The adapter proteins Appl1 (adaptor protein containing pleckstrin homology domain, phosphotyrosine domain, and leucine zipper motif 1) and Appl2 are highly homologous and involved in several signaling pathways. While previous studies have shown that Appl1 plays a pivotal role in adiponectin signaling and insulin secretion, the physiological functions of Appl2 are largely unknown. RESULTS In the present study, the role of Appl2 in sepsis shock was investigated by using Appl2 knockout (KO) mice. When challenged with lipopolysaccharides (LPS), Appl2 KO mice exhibited more severe symptoms of endotoxin shock, accompanied by increased production of proinflammatory cytokines. In comparison with the wild-type control, deletion of Appl2 led to higher levels of TNF-α and IL-1β in primary macrophages. In addition, phosphorylation of Akt and its downstream effector NF-κB was significantly enhanced. By co-immunoprecipitation, we found that Appl2 and Appl1 interacted with each other and formed a complex with PI3K regulatory subunit p85α, which is an upstream regulator of Akt. Consistent with these results, deletion of Appl1 in macrophages exhibited characteristics of reduced Akt activation and decreased the production of TNFα and IL-1β when challenged by LPS. CONCLUSIONS Results of the present study demonstrated that Appl2 is a critical negative regulator of innate immune response via inhibition of PI3K/Akt/NF-κB signaling pathway by forming a complex with Appl1 and PI3K.