In　order　to　solve　the　problems　of　rubropunctatin　insoluble　in　water　and　its　low　bioavailability,　and　explore　the　synthesis　method　of　green　silver　nanoparticles,　rubropunctatin　was　used　as　reducing　agent　and　blocking　agent,　rubropunctatin-functionalized　silver　nanoparticles　(R-AgNPs)　were　successfully　synthesized.　The　distinctive　absorption　peak　at　410　nm　confirmed　the　formation　of　R-AgNPs.　Zeta　potential　measurement　showed　excellent　stability　of　R-AgNPs　with　negative　values　of　−29.81　±　0.37　mV.　The　results　of　TEM　and　XRD　showed　that　the　prepared　R-AgNPs　were　round,　well　dispersed　and　crystallized　with　average　particle　size　of　13.54　±　0.42　nm.　FT-IR　and　XPS　studies　show　that　functional　groups　are　involved　in　R-AgNPs　synthesis.　The　antibacterial　activity　of　R-AgNPs　was　compared　with　commercial　silver　nanoparticles　(AgNPs)　by　microdilution　method.　The　results　showed　that　R-AgNPs　(MIC　7.81　μg/mL)　has　stronger　antibacterial　activity　than　commercial　AgNPs.　The　bacteria　morphology　was　observed　by　the　live　and　dead　(SYTO　9/PI)　staining　assay　and　SEM　showed　that　the　antibacterial　effect　of　R-AgNPs　was　caused　by　the　destruction　of　the　bacterial　cell　membrane.　Cytotoxicity　of　rubropunctatin-functionalized　silver　nanoparticles　and　commercial　silver　nanoparticles　on　mouse　fibroblast　3T3　cells　was　assessed　by　CCK-8　assay.　The　results　showed　that　the　toxicity　of　rubropunctatin-functionalized　silver　nanoparticles　to　3T3　cells　was　lower　than　that　of　commercial　silver　nanoparticles.　In　summary,　synthesis　of　silver　nanoparticles　using　rubropunctatin　is　a　green　synthesis　method,　and　R-AgNPs　is　a　potential　antibacterial　agent.　©　2022　by　the　authors.