This study was to determine the molecular mechanism of sodium butyrate (SB) stimulated the proliferation of bovine mammary epithelial cells (BMECs). A single factor experimental design was used to culture BMECs in DMEM/ F12 medium (including 10% fetal calf serum) containing different concentrations (0, 15, 30, 45, 60 and 75 μmol/ L) of SB, the cell activity was determined by cell count kit (CCK-8), and the optimal SB concentration was measured. Then the BMECs were cultured at control (0 μmol/ L) and optimal SB concentration, and the signaling pathway and receptor were treated by protein kinase B (AKT) blocker (AKT-IN-1), mammalian target of rapamycin (mTOR) blocker rapamycin (Rap) or small interfering RNA (siRNA) silencing G protein-coupled receptor 41 (GPR41), the proliferation and apoptosis of BMECs and the expression of genes and proteins related to GPR41 and Akt/ mTOR signaling pathways were detected. The results showed as follows: 1) compared with the control group, 60 μmol/ L SB significantly increased the cell viability of BMECs (P<0.05), however, 75 μmol/ L SB significantly inhibited the cell viability of BMECs (P<0.05); 60 μmol/ L SB significantly increased the mRNA relative expression levels of proliferating cell nuclear antigen (PCNA), cyclin A2 (CCNA2) and cyclin D1 (CCND1) (P<0.01), and significantly increased the protein relative expression levels of PCNA and cyclin A1 (CCNA1) (P<0.05). 2) Compared with the control group, 60 μmol/ L SB significantly increased the mRNA and protein relative expression levels of B-cell lymphoma 2 (BCL2) and the ratio of BCL2 / B-cell lymphoma 2 associated X protein (BAX) (P<0.05 or P< 0.01), significantly decreased the mRNA and protein relative expression levels of BAX, cysteine aspartic acid specific protease-3 (Caspase-3) and cysteine aspartic acid specific protease-9 (Caspase-9), and significantly increased the phosphorylated protein kinase (p-Akt) / Akt and phosphorylated mammalian target of rapamycin (p-mTOR) / mTOR ratios (P<0.05 or P<0.01). Moreover, the activation of Akt and mTOR signaling pathways caused by 60 μmol/ L of SB were obstructed by AKT-IN-1 (P<0.01), and the suppression of mTOR with Rap completely reversed the 60 μmol/ L SB modulated promotion of BMECs proliferation and the alteration of proliferous genes and protein expressions (P<0.05 or P<0.01), but not affected on the mRNA or proteins expression related to apoptosis and SB activated Akt signaling pathway (P>0.05). 3) Compared with the control group, 60 μmol/ L SB significantly increased the mRNA relative expression level of GPR41 (P < 0.01), and significantly increased the protein relative expression level of GPR41 (P<0.05). After silencing GPR41 with siRNA completely reversed the stimulative effect of 60 μmol/ L SB on BMECs proliferation, and mRNA or protein expression of proliferating genes and proteins associated with the Akt/ mTOR signaling pathways (P<0.05 or P<0.01). It is concluded that the proliferation of BMECs is stimulated by 60 μmol/ L SB through the GPR41 mediated Akt/ mTOR signaling pathways.