In cattle, embryo transfer into the uterine horn contralateral to the corpus luteum results in a higher incidence of pregnancy loss compared to transfer into the ipsilateral horn. We have previously reported temporal changes in the endometrial transcriptome during the estrous cycle which differ between uterine horns. The objective of this study was to compare the transcriptomic response of endometrium from the ipsilateral and contralateral horns to an elongating conceptus. Cross-bred beef heifers (n = 16) were synchronized and either used to generate day 14 conceptuses following the transfer of in vitro-produced blastocysts or to obtain day 14 endometrial explants. Conceptuses were recovered on day 14 by post-mortem uterine flushing, placed individually on top of explants collected from the ipsilateral (IPSI-D14) or the contralateral (CONTRA-D14) uterine horn of cyclic heifers, and co-cultured for 6 h. The response to a conceptus was markedly different between uterine horns, with 61 and 239 differentially expressed genes (DEGs; false discovery rate <0.05) in the ipsilateral and contralateral horns, respectively, compared to their controls. Direct comparison between IPSI-D1 and CONTRA-D14 revealed 32 DEGs, including CXCL11, CXCL10, IFIT2, RSAD2 and SAMD9. Gene Ontology analysis of these 32 genes revealed ten enriched biological processes, mainly related to immune response and response to an external stimulus. These data indicate that the endometrial response to the presence of a conceptus varies between uterine horns in the same uterus and may contribute to the higher incidence of pregnancy loss following embryo transfer to the contralateral horn.