Bruton tyrosine kinase (Btk) is not only critical for B cell development and differentiation but is also involved in the regulation of Toll-like receptor-triggered innate response of macrophages. However, whether Btk is involved in the regulation of natural killer (NK) cell innate function remains unknown. Here, we show that Btk expression is up-regulated during maturation and activation of mouse NK cells. Murine Btk(-/-) NK cells have decreased innate immune responses to the TLR3 ligand, with reduced expressions of IFN-γ, perforin, and granzyme-B and decreased cytotoxic activity. Furthermore, Btk is found to promote TLR3-triggered NK cell activation mainly by activating the NF-κB pathway. Poly(I:C)-induced NK cell-mediated acute hepatitis was observed to be attenuated in Btk(-/-) mice or the mice with in vivo administration of the Btk inhibitor. Correspondingly, liver damage was aggravated in Btk(-/-) mice after the adoptive transfer of Btk(+/+) NK cells, further indicating that Btk-mediated NK cell activation contributes to TLR3-triggered acute liver injury. Importantly, reduced TLR3-triggered activation of human NK cells was observed in Btk-deficient patients with X-linked agammaglobulinemia, as evidenced by the reduced IFN-γ, CD69, and CD107a expression and cytotoxic activity. These results indicate that Btk is required for activation of NK cells, thus providing insight into the physiological significance of Btk in the regulation of immune cell functions and innate inflammatory response.