TLR4 was the first TLR family member identified in mammals and is responsible for the activation of the immune response by bacterial LPS. Later, MD1 and RP105 were shown to form complexes that directly interact with the MD2-TLR4 complex, acting as physiological negative regulators of LPS signaling. Despite the general conservation of various TLR families from fish to mammals, several differences can be appreciated, such as the high tolerance of fish to LPS, the absence of the crucial accessory molecules Md2 and Cd14 for Tlr4 signaling in fish, the absence of Tlr4 in some fish species, and the confirmation that LPS does not signal through Tlr4 in zebrafish. The present study has identified the Rp105 and Md1 homologs in zebrafish, confirming (i) Rp105 and Tlr4 evolved from a common ancestor before the divergence between fish and tetrapods and (ii) the presence of Md1 in teleost fish and the lack of Md2, suggesting that the divergence of these accessory molecules occurred in the tetrapod lineage. Biochemical and functional studies indicate that Md1 binds both Rp105 and Tlr4 in zebrafish. Genetic inhibition of zebrafish Md1 and Rp105 reveals that Md1 or Rp105 deficiency impairs the expression of genes encoding pro-inflammatory and antiviral molecules, leading to increased susceptibility to viral infection. These results shed light on the evolutionary history of Md1 and Rp105 and uncover a previously unappreciated function of these molecules in the regulation of innate immunity.,Copyright © 2015 Elsevier Ltd. All rights reserved.