Lipopolysaccharide (LPS) is a cell wall component of Gram negative bacteria. LPS activates immune cells and triggers production of proinflammatory cytokines and other mediators through Toll-like receptor (TLR) 4. Src homology (SH) 2 domain-containing inositol-5-phosphatase 1 (SHIP1) plays important roles in negatively regulating the activation of immune cells primarily via phosphoinositide 3-kinase (PI3K) pathway by catalyzing PI3K product PtdIns-3,4,5P3 into PtdIns-3,4P2. However, little is known about the role of SHIP1 in TLR4-mediated LPS responses. We investigated the role of SHIP1 in LPS-induced MAPKs activation and IkappaB-alpha degradation by overexpression and RNA interfering experiments. SHIP1 becomes tyrosine phosphorylated and membrane translocated upon LPS stimulation in RAW264.7 macrophages. Stable SHIP1 transfection inhibits LPS-induced TNF-alpha and IL-6 production by 60% and 70% respectively. Transient SHIP1 transfection inhibits LPS-induced phospharylation of extracellular signal-regulated kinase (ERK1/2), p38, c-Jun NH2-terminal kinase (JNK) and degradation of IkappaB-alpha by about 20%, 50%, 40% and 40% respectively at the time point of 20 min after LPS stimulation. Disruption of SHIP1 phosphatase activity enhances LPS-induced activation of Akt, a downstream molecule of PI3K pathway, but fails to reverse SHIP1 mediated inhibition of LPS-induced activation of MAPKs and degradation of IkappaB-alpha. Consistently, LY 294002 and Wortmannin sufficiently inhibit LPS-induced Akt activation but can not diminish SHIP1 mediated inhibition of LPS-induced activation of MAPKs and degradation of IkappaB-alpha. On the contrary, SHIP1-specific RNA interfering inhibits SHIP1 expression and enhances LPS-induced phospharylation of ERK1/2, p38, JNK and degradation of IkappaB-alpha by about 2.6, 2.6, 3.0 and 8.0-fold respectively at the time point of 20 min after LPS stimulation. Even in the presence of LY 294002, SHIP1 RNA interfering could increase LPS-induced IL-6 production. Furthermore, SHIP1 overexpression nearly completely abolished TLR4 reconstitution conferred LPS responsiveness in COS7 cells. These results demonstrated that SHIP1 negatively regulates LPS responses in macrophage via phosphatase activity and PI3K-independent mechanism.