TLR agonists such as lipopolysaccharide (LPS) and poly(I:C) induce expression of type I interferons such as IFN-alpha and IFN-beta by macrophages. To examine the role of IFN-β in the induction of IFN-stimulated genes (ISGs) by LPS, we compared the ability of LPS to induce ISGF3 activity and ISG expression in bone marrow-derived macrophages from wild-type (WT) and IFN-β gene knockout (Ifnb1-/-) mice. We found that LPS treatment activated ISGF3 and induced expression of ISGs such as Oas1, Mx1, Ddx58 (RIG-I) and Ifih1 (MDA5) in WT macrophages, but not in macrophages derived from Ifnb1-/- mice or IFN-a/β receptor gene knockout (Ifnar1-/-) mice. The inability of LPS to induce activation of ISGF3 and ISG expression in Ifnb1-/- macrophages correlated with the failure of LPS to induce activation of STAT1 and STAT2 in these cells. Consistent with these findings, LPS treatment also failed to induce ISG expression in bone marrow-derived macrophages from Stat2 knockout mice. Although activation of ISGF3 and induction of ISG expression by LPS was abrogated in Ifnb1-/- and Ifnar1-/- macrophages, activation of NF-kB and induction of NF-kB responsive genes such as Tnf (TNF-a) and Il1b (IL-1β) were not affected by deletion of either the IFN-β or IFN-aR1 genes. These findings demonstrate that induction of ISGF3 activity and ISG expression by LPS is critically dependent on intermediate production of IFN-β and autocrine signaling through type I IFN receptors.