A previous study showed that Peroxisome Proliferator-Activated Receptor β/δ (PPARβ/δ) activation inhibits glucose uptake in bovine aortic endothelial cells. We hypothesize that inhibition of PPARβ/δ can increase glucose uptake in immortalized bovine mammary alveolar cells (MACT) and increases lactose synthesis. To test our hypothesis, we treated MACT cells with PPARβ/δ synthetic agonist (GW501516) and antagonist (GSK3787) and assessed PPARβ/δ activation, live and dead cell count, and glucose uptake. MACT cells plated at 10,000 cells/well in 96 well plates were transfected with a PPAR Response Element (PPRE X3-TK-luc) plasmid using 0.3 μL/well of TransIT-X2 Dynamic Delivery System (Mirus) in Opti-MEM media without fetal bovine serum (FBS). Cells were treated 24h after transfection in triplicates with 10 and 1,000 nM of GW501516, GSK3787, or a 1:1 combination of them plus ethanol as control in high-glucose DMEM medium with 10% FBS. After 24h of treatment, a nuclear staining (NucBlue Live) was added and 2 images/well were obtained using an inverted fluorescent microscope (DMI6000B, Leica Microsystems, Germany). Luciferase activity was measured via a luminometer and normalized by the number of viable cells measured using CellProfiler software. Glucose concentration in the medium was measured using a Blood Glucose Meter kit (Safeway) and glucose uptake/viable cell was estimated. Data were analyzed using GLIMMIX of SAS. Significance was declared with P < 0.05. More than 2-fold increase of luciferase compared with control (P < 0.001) was observed with both doses of GW501516 and a dose-dependent decrease of luciferase was observed with GSK3787. The number of viable cells was negatively affected by 10 nM of GSK3787 (P < 0.05) but was positively affected with 1,000 nM of GSK3787 (16,990 vs. 20,966 ± 521 cells/well). No effect on cell viability was observed with GW501516. There was a tendency (P = 0.06) for an overall effect of treatments on glucose uptake. Cells treated with GW501516 had a lower glucose uptake (7.2 vs. 10.4 ± 1.6 ng/cell) compared with cells treated with GSK3787. In conclusion, the use of 1,000 nM of GSK3787 successfully inhibited PPARβ/δ activity and increased glucose uptake in MACT cells. It remains to be determined if the increase in glucose uptake observed results in higher lactose synthesis.