Of 10 mammalian secreted phospholipase A₂ (sPLA₂) enzymes identified to date, group V and X sPLA₂s, which are two potent plasma membrane-acting sPLA₂s, are capable of preventing host cells from being infected with adenovirus, and this anti-viral action depends on the conversion of phosphatidylcholine (PC) to lysophosphatidylcholine (LPC) in the host cell membrane. Here, we show that human group III sPLA₂, which is structurally more similar to bee venom PLA₂ than to other mammalian sPLA₂s, also has the capacity to inhibit adenovirus infection into host cells. Mass spectrometry (MS) demonstrated that group III sPLA₂ hydrolyzes particular molecular species of PC to generate LPC in human bronchial epithelial cells. Remarkably, in addition to the catalytically active sPLA₂ domain, the N-terminal, but not C-terminal, domain unique to this enzyme was required for the anti-adenovirus effect. To our knowledge, this is the first demonstration that the biological action of group III sPLA₂ depends on its N-terminal domain. Finally, our MS analysis provided additional and novel evidence that group III, V and X sPLA₂s target distinct phospholipid molecular species in cellular membranes.