The physiological changes associated with the varying hormonal environment throughout the oestrous cycle are linked to the different functions the uterus needs to fulfil. The aim of the present study was to generate global gene expression profiles for the equine uterus during oestrus and Day 5 of dioestrus. To achieve this, samples were collected from five horses during oestrus (follicle >35 mm in diameter) and dioestrus (5 days after ovulation) and analysed using high-throughput RNA sequencing techniques (RNA-Seq). Differentially expressed genes between the two cycle stages were further investigated using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. The expression of 1577 genes was found to be significantly upregulated during oestrus, whereas 1864 genes were expressed at significantly higher levels in dioestrus. Most genes upregulated during oestrus were associated with the extracellular matrix, signal interaction and transduction, cell communication or immune function, whereas genes expressed at higher levels in early dioestrus were most commonly associated with metabolic or transport functions, correlating well with the physiological functions of the uterus. These results allow for a more complete understanding of the hormonal influence on gene expression in the equine uterus by functional analysis of up- and downregulated genes in oestrus and dioestrus, respectively. In addition, a valuable baseline is provided for further research, including analyses of changes associated with uterine inflammation.