Penicillin　V　is　a　bacteriolytic　β-lactam　antibiotic　drug.　In　the　present　work,　we　investigated　the　inhibitory　effect　of　Penicillin　V　on　the　activity　of　mushroom　tyrosinase　for　the　first　time.　The　molecular　mechanism　for　the　inhibition　of　tyrosinase　by　Penicillin　V　was　investigated　by　means　of　kinetics　analysis,　fluorescence　quenching　and　molecular　docking　techniques.　The　results　showed　that　Penicillin　V　could　inhibit　both　monophenolase　and　diphenolase　activities　with　IC50　of　16.6　±　0.5　and　11.0　±　0.2 mmol/L,　respectively.　The　inhibitory　type　of　Penicillin　V　on　mushroom　was　mixed　type,　and　the　values　of　KI　and　KIS　were　13.46　and　17.26 mmol/L,　respectively.　The　fluorescence　quenching　and　molecular　docking　showed　that　Penicillin　V　could　form　static　interaction　near　the　catalytic　pocket　of　the　enzyme　to　hinder　the　transportation　of　substrate　to　the　active　site,　as　well　as　reduce　the　copper　plasticity　for　catalysis.　Our　results　contributed　to　the　usage　of　Penicillin　V　as　a　novel　tyrosinase　inhibitor　with　dual　effect　in　field　of　antimicrobial　and　food　preservation　and　could　also　provide　guidance　for　the　design　of　novel　tyrosinase　inhibitors.　©　2019,　Springer　Nature　B.V.