Both form and concentration of supplemental Zn has been shown to impact milk production and mammary health in dairy cattle. However, the physiological mechanisms by which these effects are produced remain to be fully elucidated. One potential route is by direct effect on mammary epithelial cells (MEC). Zinc is known to act as a ligand for GPR39, a G protein-coupled receptor expressed in a variety of cell lines and tissues, where it promotes cell survival and proliferation by a G_αq pathway characterized by intracellular Ca⁺⁺ release followed by phosphorylation of kinases including ERK and AKT. The objective of this study was to characterize the presence and activity of GPR39 in an immortalized bovine MEC line (MAC-T). Using RT-qPCR, GPR39 was found to be expressed in a variety of bovine tissues as well as in MAC-T cells. Two siRNA constructs (siGRP39a and siGPR39b) were designed and utilized in vitro for the knockdown of GPR39 expression in MAC-T cells. Cells were cultured on 12-well plates, transfected with siGRP39a, siGPR39b, or a universal negative control (siCON) 24 h before subsequent treatments. Cells were then treated for 10 min with either a Zn-free physiological saline solution (0 Zn), or 100 µM Zn (100 Zn), and after another 10 min, cells were harvested for RNA and protein. Transcript abundance was determined by RT-qPCR and protein phosphorylation by Western blot. There was a tendency for 100 Zn to increase GPR39 mRNA abundance compared with 0 Zn in siCON cells (P = 0.096). In 100 Zn cells, transcript abundance of GPR39 was reduced 63% by siGPR39a (P = 0.02) and 57% by siGPR39b (P = 0.04). No effects of GPR39 knockdown, Zn treatment, or their interaction were observed on phosphorylation of AKT or ERK, 2 common intermediates of G_αq signaling. In summary, extracellular Zn was not observed to activate G_αq signaling in MAC-T cells regardless of GPR39 expression.