Defensins are important molecules in the innate immune system that eliminate infectious microbes. They also exhibit cytotoxicity against host cells in higher concentrations. The mechanisms by which hosts protect their own cells from cytotoxicity of defensins have been poorly understood. We found that the cytotoxicity of human β-defensin 3 (hBD3) against lung epithelial cells was dose-dependently attenuated by pulmonary surfactant protein A (SP-A), a collectin implicated in host defense and regulation of inflammatory responses in the lung. The direct interaction between SP-A and hBD3 may be an important factor in decreasing this cytotoxicity because preincubation of epithelial cells with SP-A did not affect the cytotoxicity. Consistent with in vitro analysis, intratracheal administration of hBD3 to SP-A(-/-) mice resulted in more severe tissue damage compared with that in WT mice. These data indicate that SP-A protects lung epithelium from tissue injury caused by hBD3. Furthermore, we found that the functional region of SP-A lies within Tyr(161)-Lys(201). Synthetic peptide corresponding to this region, tentatively called SP-A Y161-G200, also inhibited cytotoxicity of hBD3 in a dose-dependent manner. The SP-A Y161-G200 is a candidate as a therapeutic reagent that prevents tissue injury during inflammation.