The　severe　acute　respiratory　syndrome　coronavirus-2　(SARS-CoV-2)　helicase　NSP13　plays　a　conserved　role　in　the　replication　of　coronaviruses　and　has　been　identified　as　an　ideal　target　for　the　development　of　antiviral　drugs　against　SARS-CoV-2.　Here,　we　identify　a　novel　NSP13　helicase　inhibitor　punicalagin　(PUG)　through　high-throughput　screening.　Surface　plasmon　resonance　(SPR)-based　analysis　and　molecular　docking　calculation　reveal　that　PUG　directly　binds　NSP13　on　the　interface　of　domains　1A　and　2A,　with　a　KD　value　of　21.6　nM.　Further　biochemical　and　structural　analyses　suggest　that　PUG　inhibits　NSP13　on　ATP　hydrolysis　and　prevents　it　binding　to　DNA　substrates.　Finally,　the　antiviral　studies　show　that　PUG　effectively　suppresses　the　SARS-CoV-2　replication　in　A549-ACE2　and　Vero　cells,　with　EC50　values　of　347　nM　and　196　nM,　respectively.　Our　work　demonstrates　the　potential　application　of　PUG　in　the　treatment　of　coronavirus　disease　2019　(COVID-19)　and　identifies　an　allosteric　inhibition　mechanism　for　future　drug　design　targeting　the　viral　helicases.　©　2022　Elsevier　B.V.