sPLA₂ (secretory phospholipase A₂) enzymes have been implicated in various biological events, yet their precise physiological functions remain largely unresolved. In the present study we show that group V and X sPLA₂s, which are two potent plasma membrane-acting sPLA₂s, are capable of preventing host cells from being infected with an adenovirus. Bronchial epithelial cells and lung fibroblasts pre-expressing group V and X sPLA₂s showed marked resistance to adenovirus-mediated gene delivery in a manner dependent on their catalytic activity. Although adenovirus particles were insensitive to recombinant group V and X sPLA₂s, direct addition of these enzymes to 293A cells suppressed both number and size of adenovirus plaque formation. Group V and X sPLA₂s retarded the entry of adenovirus into endosomes. Moreover, adenoviral infection was suppressed by LPC (lysophosphatidylcholine), a membrane-hydrolytic product of these sPLA₂s. Thus hydrolysis of the plasma membrane by these sPLA₂s may eventually lead to the protection of host cells from adenovirus entry. Given that group V and X sPLA₂s are expressed in human airway epithelium and macrophages and that the expression of endogenous group V sPLA₂ is upregulated by virus-related stimuli in these cells, our present results raise the possibility that group V and X sPLA₂s may play a role in innate immunity against adenoviral infection in the respiratory tract.