Conceptus elongation coincides with one of the periods of greatest pregnancy loss in cattle and is characterized by rapid trophectoderm expansion, commencing ~ Day 13 of pregnancy, i.e. before maternal pregnancy recognition. The process has yet to be recapitulated in vitro and does not occur in the absence of uterine gland secretions in vivo. Moreover, conceptus elongation rates are positively correlated to systemic progesterone in maternal circulation. It is, therefore, a maternally-driven and progesterone-correlated developmental phenomenon. This study aimed to comprehensively characterize the biochemical composition of the uterine luminal fluid on Days 12–14 – the elongation-initiation window – in heifers with normal vs. high progesterone, to identify molecules potentially involved in conceptus elongation initiation. Specifically, nucleotide, vitamin, cofactor, xenobiotic, peptide, and energy metabolite profiles of uterine luminal fluid were examined. A total of 59 metabolites were identified, of which 6 and 3 displayed a respective progesterone and day effect, whereas 16 exhibited a day by progesterone interaction, of which 8 were nucleotide metabolites. Corresponding pathway enrichment analysis revealed that pyridoxal, ascorbate, tricarboxylic acid, purine, and pyrimidine metabolism are of likely importance to to conceptus elongation initiation. Moreover, progesterone reduced total metabolite abundance on Day 12 and may alter the uterine microbiome.