Glucose utilisation for lactose synthesis in the mammary gland involves expression of a large number of genes whose nutritional regulation remains poorly defined. In this study, the effect of long term under- and over-feeding on the expression of genes [glucose transporter 1: GLUT1, glucose transporter 3: GLUT3, Sodium glucose contransporter 1: SGLT1, two isoforms of β- (1,4) galactosyltransferase: β- (1,4) GAT1, β- (1,4) GAT3 and α-lactalbumin: LALBA] related to glucose metabolism in sheep mammary tissue (MT) was examined. Twenty-four lactating dairy sheep were divided into three homogenous sub-groups and fed the same ration in quantities which met 70% (underfeeding), 100% (control) and 130% (overfeeding) of their energy and crude protein requirements. The results showed a significant reduction on mRNA of GLUT1 and LALBA gene in the MT of underfed sheep, compared with the respective controls and overfed and a significant reduction on mRNA level of SGLT1 and β- (1,4) GAT1 in the MT of underfed sheep, compared with the overfed ones. A significant increase in the GLUT3 mRNA accumulation in the MT of both under- and over- fed sheep was found. Additionally, a trend of reduction on β- (1,4) GAT3 mRNA level in the MT of the underfed sheep, compared with the overfed, was observed. A close positive relationship was obtained between the mRNA transcripts accumulation of GLUT1, SGLT1, β- (1,4) GAT1 and LALBA gene with the milk lactose content and milk lactose yield respectively. In conclusion, feeding level and consequently nutrient availability, may affect glucose uptake and utilisation in sheep MT by altering the GLUT1, GLUT3, SGLT1, β- (1,4) GAT1 and LALBA gene expression involved in their metabolic pathways.