The experiment was designed to evaluate the effect of sodium selenite (Na₂SeO₃) on the proliferation of bovine mammary gland epithelial cells (BMECs), and to assess the underlying mechanisms. The addition of Na₂SeO₃ at doses ranging from 18 to 140 nM stimulated BMEC proliferation. Nevertheless, high doses of Na₂SeO₃ (> 70 nM) did not further stimulate BMEC proliferation compared to 35 nM Na₂SeO₃. Therefore, the addition of 35 nM Na₂SeO₃ increased the proportion of epithelial cells undergoing DNA replication, mRNA expression of proliferating cell nuclear antigen (PCNA), Cyclin A2 (CCNA2) and Cyclin D1 (CCND1), as well as protein expression of PCNA and Cyclin A1. Transcription and translation of the B-cell lymphoma 2 (BCL2) gene and the ratio of BCL2 to BCL2-associated X 4 (BAX4) genes were significantly elevated, while mRNA and protein expressions of BAX, caspase 3 (CASP3) and caspase 9 (CASP9) genes were significantly reduced as a result of 35 nM Na₂SeO₃ supplementation. Additionally, both the Akt and mTOR signalling pathways were activated by 35 nM Na₂SeO₃. On the other hand, the stimulation of BMEC proliferation, altered expression of proliferative and apoptotic genes and proteins, as well as mTOR signalling pathway activation caused by 35 nM Na₂SeO₃ addition was suppressed by the Akt inhibitor (AKT-IN-1). Likewise, rapamycin-mediated suppression of mTOR completely reversed the 35 nM Na2SeO₃-stimulated BMEC proliferation and alteration of proliferous gene and protein expressions, without affecting mRNA or protein expression of genes related to apoptosis and the Na₂SeO₃-activated Akt signalling pathway. In conclusion, the results implicated that the proliferation of BMECs was stimulated by 35 nM Na₂SeO₃ through the Akt-mTOR signalling pathway.