The equine oviduct plays a pivotal role in providing the optimal microenvironment for early embryonic development, but little is known about the protein composition of the oviducal fluid in the horse. The aim of the present study was to provide a large-scale identification of proteins in equine oviducal fluid and to determine the effects of ovulation and pregnancy. Four days after ovulation, the oviducts ipsilateral and contralateral to the ovulation side were collected from five pregnant and five non-pregnant mares. Identification and relative quantification of proteins in the oviducal fluid of the four groups was achieved by isobaric tags for relative and absolute quantification (iTRAQ) labelling and HPLC-tandem mass spectrometry. The presence of an embryo in the ipsilateral oviducal fluid of pregnant mares induced upregulation of 11 and downregulation of two proteins compared with the contralateral side, and upregulation of 19 proteins compared with the ipsilateral side of non-pregnant mares. Several of these upregulated proteins are related to early pregnancy in other species. The present study represents the first high-throughput identification of proteins in the oviducal fluid of the mare. The results support the hypothesis that the equine embryo interacts with the oviduct, affecting the maternal secretion pattern of proteins involved in pregnancy-related pathways.