Toll like receptors (TLRs) play a critical role in the immediate detection and control of infection in mammals and are important for initiating a series of signaling events which result in proinflammatory gene expression. Many of these receptors are type I transmembrane proteins that belong to the IL-1 receptor family. There are 13 TLRs already identified in mammals, 10 in humans, 10 in bovine and 13 in mice. The TLRs main functions include secretion of type I interferon, proinflamatory cytokines, chemokines and expression of co-stimulatory molecules. The aim of this study was to verify intracellular perforin and IFN-γ production, granzyme and perforin transcription and extra cellular IFN-γ secretion in bovine NK cells treated with TLRs ligands such as TLR2, TLR3, TLR4, TLR5 and TLR9. Our preliminary results demonstrated that flagellin (6/6) and PPD (5/6) are the most inducers of intracellular IFN-γ production followed by LPS (4/6) and CpG (4/6). When we analyzed the intracellular perforin production we verified an increase in the cells treated with CpG (4/6) or LPS (3/6). The IFN-γ secretion in the supernatant was analized by a capture ELISA. The Poly I:C had the bigger impact in IFN-γ secretion followed by CpG and LPS which caused a small increase, while PPD and flagellin did not have any effect in IFN-γ secretion. These results suggest that bovine NK cells might be activated by these TLRs ligants, otherwise further studies are needed to elucidate this hypothesis.