Toll-like receptors (TLRs) have been shown to play a crucial role in host defense against pathogenic microbes in innate immunity in mammals. Recent genome-wide analyses have suggested that TLRs or related genes are conserved in the genome of non-mammalian organisms such as fishes, cyclostomes, ascidians, cephalochordates, sea urchins, and hydras. However, neither active forms nor functions of authentic invertebrate TLRs had been elucidated. Quite recently, we verified the structures, localization, ligand recognition, activities, and inflammatory cytokine production of two TLRs in the ascidian Ciona intestinalis, designated Ci-TLR1 and Ci-TLR2. Both Ci-TLRs possess a unique structural organization, with moderate sequence similarity to functionally characterized vertebrate TLRs, and are expressed predominantly in the stomach and intestine as well as in hemocytes. Unlike vertebrate TLRs, Ci-TLR1 and Ci-TLR2 are present in both the plasma membrane and endosomes. Furthermore, both Ci-TLR1 and Ci-TLR2 stimulate NF-κ;B induction in response to multiple pathogenic ligands that are differentially recognized by respective vertebrate TLRs. Pathogenic ligands that stimulate the Ci-TLRs also induce the expression of Ci-TNFα in the intestine and stomach, where the Ci-TLRs are abundantly expressed. These data reveal the conservation of the TLR-triggered innate immune system in C. intestinalis, and both common and unique biological and immunological functions of the Ci-TLRs. Based on the latest findings, we review recent advances in studies of TLRs or related receptors in fish, cyclostomes, deuterostome invertebrates, and hydra, and also the significance of studies of lower organism TLRs.