The Toll-like receptors (TLRs) are a family of pathogen recognition receptors that alert the host to the presence of microbial challenge. Each TLR responds to a specific microbial associated ligand. For example, TLR4 is activated by lipopolysaccharide (LPS), whereas TLR9 responds to microbial DNA (CpGs). In this report signal transduction responses of bovine monocytes to stimulation with LPS and CpG are described through a bovine-specific peptide array. In addition to confirming activation of the defined TLR pathway in bovine cells, unique phosphorylation events not previously attributed to TLR signaling are described and validated. For example, array data predicts phosphorylation of Tyr40 of Etk in response to LPS, but not CpG, stimulation as well as the activation of oxidative burst in CpG, but not LPS. This investigation confirms interspecies conservation of the TLR pathway in bovine as well as providing insight into the complexity and mechanisms of TLR signaling.