Objective To explore the proliferation-promoting effect of bovine mammary gland epithelial cells (BMECs) co-cultured with umbilical cord mesenchymal stem cells (UC-MSCs) in serum-free culture mediuum. Methods Bovine UC-MSCs and BMECs were selected for co-culturing in direct or indirect contact. In the direct contact culture groups, UC-MSCs and BMECs were co-cultured at concentration ratios of 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, and 1:10, respectively. In the indirect contact culture group, the supernatant of UC-MSCs was used as the conditioned medium to re-suspend BMECs. In the control groups, UC-MSCs and BMECs were cultured alone. The cell growth status in each group was observed at 0, 4, 8, 12, 24, 36, 48, 60, 72 h after culture, and cell proliferation was detected by cell counting kit-8 (CCK-8) assay. Results At 48 h, the optical density of the conditioned medium-BMECs group was significantly higher compared with the control groups (P<0.05). Meanwhile, the optical density in the direct contact group at a concentration ratio of 1:2 reached the peak, which was extremely significantly higher compared with the control groups (P<0.01) and significantly higher compared with the other direct contact culture groups and the conditioned medium-BMECs group (P<0.05). Conclusions Co-culture of UC-MSCs and BMECs in serum-free culture medium is capable to promote the proliferation of BMECs, and the co-culture by cell-to-cell contact has a better effect. The optimal concentration ratio of UC-MSCs to BMECs is 1:2, and the optimal culture time is 48 h.