Heparin-binding Epidermal Growth Factor-like Growth Factor (HBEGF) is a member of the epidermal growth factor (EGF) family. As a ligand for the EGF receptor (EGFR), HBEGF can bind to both 170 kDa EGFR and 180 kDa ErbB4, and through heterodimerization, ErbB2. HBEGF protein involves in several processes, including epidermal growth factor receptor signaling pathway，phosphatidylinositol 3-kinase/protein kinase B signal transduction and wound healing. To test the effect of HBEGF on cell proliferation, BALB/3T3 cells were seeded into triplicate wells of 96-well plates and allowed to attach, replaced with various concentrations of recombinant human HBEGF. After incubated for 72h, cells were observed by inverted microscope and cell proliferation was measured by Cell Counting Kit-8 (CCK-8). Briefly, 10 µl of CCK-8 solution was added to each well of the plate, then the absorbance at 450 nm was measured using a microplate reader after incubating the plate for 1-4 hours at 37 ℃. Cell viability was assessed by CCK-8 assay after incubation with recombinant human HBEGF for 72h. The result was shown in Figure 1. It was obvious that HBEGF significantly increased cell viability of BALB/3T3 cells. The ED50 of recombinant human HBEGF is 1.033μg/ml.