Growth hormone (GH) may exert its function through direct activation of the the growth hormone receptor (GHR) in mammary gland or through stimulating insulin-like growth factor-I (IGF-I). We hypothesized that the reduction of milk yield caused in part by apoptosis of mammary epithelial cells depends on the ratio of GH and IGF-I which could be influenced by nutrition, however, evidence to support this suggestion is lacking. Therefore, the purpose of this study was to investigate the effects of GH and IGF-I alone and in combination on proliferation and apoptosis of bovine mammary epithelial cells cultured in vitro. Mammary epithelial cells isolated from bovine mammary tissue were identified by cytomorphology, immunocytochemistry and specific gene expression. Subcultured and purified cells were treated with GH (100 ng/mL) and IGF-I (100 ng/mL) alone or a combination of GH and IGF-I (each 100 ng/mL) in growth medium without fetal calf serum for 24 h. The mRNA abundance was quantified by RT-qPCR, cell proliferation was determined with a CCK-8 kit, and apoptosis was identified by an FITC-Annexin V/PI kit. Statistical analysis was carried out by using ANOVA procedure of SAS 9.1 software with Duncan’s multiple-range test. Cell proliferation was not enhanced by adding GH alone (P > 0.05) while it was promoted by IGF-I at about 6 h (P < 0.05). The combination of GH and IGF-I increased cell proliferation from 4 to 24 h (P < 0.05), which had no significant difference with IGF-I group (P > 0.05); The supplementation of GH or IGF-I individually had no significant effect on the early apoptosis rates of cells (P > 0.05), however, the late (P < 0.01) and total apoptosis rates (P < 0.05) were reduced. The late and total cell apoptosis rates were decreased by adding GH and IGF-I together (P < 0.01). In addition, adding GH or IGF-I alone tended to decreased the mRNA abundance of IGFBP-5 (P < 0.1) compared with the control, and the inhibitory effect was strongest when GH was combined with IGF-I (P < 0.05). This study demonstrated that GH and IGF-I can regulate the number and activity of bovine mammary epithelial cell through promoting cell proliferation and inhibiting cell apoptosis to stimulate lactation.