Abstract: Objective To investigate the effect of exosome secreted by mouse fibroblast cell line 3T3 on the proliferation of mouse breast cancer cells 4T1, and to explore the potential underlying mechanism. Methods The exosome was obtained and purified by PureExo Exosome kit from mouse fibroblast cell line 3T3 cultural supernatant, and 4T1 cells were cultured and incubated with different doses of exosome for indicated time. Then CCK8 assay and BrdU/PI incorporation were performed to measure the proliferation and cell cycle of 4T1 cells. Western blot and qPCR were used to detect and analyze the expression of human epidermal growth factor receptor-2(HER2) and the activity of its downstream PI3K/AKT signal pathway. The monoclonal antibody of HER2, Herceptin, was used to determine whether exosome regulated the proliferation of 4T1 via HER2. Results The CCK8 assay showed at OD450 nm, the absorbency of exosome group was obvious higher than that in control group (P<0.05). The 4T1 cells treated by exosome also had faster proliferation and more aggressive cell cycle compared with untreated cells in control group. Meantime, exosome also increased the expression of HER2 determined by Western blot and qPCR, with elevating the expression of p-AKT. Meanwhile, exosome enhanced the sensitivity of 4T1 cells to Herceptin treatment. Conclusion The exosome extracted from mouse fibroblast cell line 3T3 could promote the proliferation and cell cycle progress of mouse breast cancer cells 4T1, and HER2 and its downstream PI3K/AKT signal pathway might play an important role in this regulation.