Activation of innate immune receptors by exogenous substances is crucial for the detection of microbial pathogens and a subsequent inflammatory response. The inflammatory response to microbial lipopolysaccharide via Toll-like receptor 4 (TLR4) is facilitated by soluble accessory proteins, but the role of such proteins in the activation of other pathogen recognition receptors for microbial nucleic acid is not well understood. Here we demonstrate that RNase4 and RNase5 purified from bovine milk bind to Salmonella typhimurium DNA and stimulate pro-inflammatory responses induced by nucleic acid mimetics and S. typhimurium DNA in an established mouse macrophage cell culture model, RAW264.7, as well as in primary bovine mammary epithelial cells. RNase4 and 5 also modulated pro-inflammatory signalling in response to nucleic acids in bovine peripheral blood mononuclear cells, although producing a distinct response. These results support a role for RNase4 and RNase5 in mediating inflammatory signals in both immune and epithelial cells, involving mechanisms that are cell-type specific.