Regulation of cell proliferation is essential for controlling organ size and maintenance of tissue homeostasis in adult organisms. Growth inhibition, termed as “contact inhibition”, in a cell-density dependent manner was common in vitro culture. In this study, the effect of glycosphingolipid GM1 on the contact inhibition of human mammary epithelial cell lines MCF-10A, human breast cancer cell lines BT-549 and SK-BR-3 were investigated. Changes of cell proliferation of MCF-10A, BT-549 and SK-BR-3 cells at low and high cell density was explored. Expression of GM1 in different cell density was detected by flow cytometry. Exogenous GM1 at different cell density was added to explore the effect on cell proliferation. B3GALT4, the GM1 synthase, was knocked down or overexpressed in BT-549 and SK-BR-3 cells by using lentiviral vectors. Then, proliferating ability were tested by cell counting and related pathways was assayed by Western blot, in stable-transfected cell lines. Results showed cell proliferation was inhibited and GM1 expression increased at low and high cell density compared with normal density. Exogenous addition of GM1 at low and high density cells inhibited cell growth, but it had no influence on cell growth at normal density. Down regulation of GM1 promoted cell proliferation at low and high cell density, and overexpression of GM1 had the opposite effect. Together, these results indicate that GM1 inhibit MCF-10A, BT-549 and SK-BR-3 cells proliferation at low and high cell density in vitro, which may potentially provide the experimental basis for further research on its molecular mechanisms.