Pseudostellaria　heterophylla　protein　hydrolysate　has　been　reported　to　be　a　potential　nutraceutical　supplement　to　strengthen　the　immune　system.　A　specific　peptide　from　P.　heterophylla　protein　hydrolysate　with　proliferation　promotion　effect　on　spleen　lymphocytes　was　purified　by　RP-HPLC.　The　amino　acid　sequence　of　the　peptide　was　determined　to　be　RGPPP,　with　molecular　weight　of　522.29　Da.　100　μg/mL　of　RP-5　achieved　the　highest　stimulation　index　of　1.27　on　spleen　lymphocytes　and　activated　RAW264.7　cells　resulting　in　up-regulating　tumor　necrosis　factor-α　(TNF-α)　secretion　to　827.24　pg/mL　and　elevating　pinocytosis　rate　to　123.81%.　Results　of　western-blot　and　siRNA　experiments　indicated　that　RP-5　activated　nuclear　factor-κB　(NF-κB)　signaling　pathway　through　combining　to　TLR2　expressed　on　RAW264.7　cells　surface.　Furthermore,　TNF-α　secretion　increased　by　RP-5　could　be　decreased　to　401.68　pg/mL　by　pyrrolidine　dithiocarbamate　or　BAY11-7082.　These　findings　further　the　research　progress　of　peptide　with　immunomodulatory　activity　and　its　action　mechanism.　©　2020　Elsevier　Ltd