Epidermal growth factor (EGF) is a growth factor that stimulates cell growth, proliferation, and differentiation by binding to its receptor EGFR. To test the effect of EGF on cell proliferation of 3T3 fibroblasts , Balb/c 3T3 cells were seeded into triplicate wells of 96-well plates at a density of 8, 000 cells/well and allowed to attach overnight, then the medium was replaced with 0.4% FBS standard DMEM prior to the addition of various concentrations of recombinant human EGF. After incubated for 48h, 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 measure the absorbance at 450 nm using a microplate reader after incubating the plate for 1-4 hours at 37 °C. Cell proliferation of Balb/c 3T3 cells after incubation with rhEGF for 48h observed by inverted microscope was shown in Figure 1. The dose-effect curve of rhEGF was shown in Figure2. It was obvious that rhEGF significantly promoted cell proliferation of 3T3 cells. The ED50 for this effect is typically 4.2 ng/ml.