The potential for dietary supplementation with n-3 polyunsaturated fatty acids (n-3 PUFA) to improve reproductive efficiency in cattle has received much interest. The mechanisms by which n-3 PUFA may affect physiological and biochemical processes in key reproductive tissues are likely to be mediated by significant alterations in gene expression. The objective of this study was to examine the effects of dietary n-3 PUFA supplementation on global uterine endometrial gene expression in cattle. Beef heifers were supplemented with a rumen protected source of either a saturated fatty acid (CON; palmitic acid) or high n-3 PUFA (n-3 PUFA; 275 g) diet per animal per day for 45 days and global gene expression was determined in uterine endometrial tissue using an Affymetrix oligonucleotide bovine array. A total of 1,807 (946 up- and 861 downregulated) genes were differentially expressed following n-3 PUFA supplementation. Dietary n-3 PUFA altered numerous cellular processes potentially important in the control of reproduction in cattle. These included prostaglandin biosynthesis, steroidogenesis and transcriptional regulation, while effects on genes involved in maternal immune response and tissue remodeling were also observed. This study provides new insights into the effects of n-3 PUFA supplementation on the regulation of gene expression in the bovine uterus.