Pregnancy establishment in cattle is contingent on conceptus elongation-a fundamental developmental event coinciding with the time during which most pregnancies fail. Elongation in vivo is directly driven by uterine secretions, indirectly influenced by systemic progesterone concentrations, and has yet to be recapitulated in vitro. To better understand the microenvironment evolved to facilitate this phenomenon, the amino acid and carbohydrate composition of uterine fluid was interrogated using high-throughput metabolomics on days 12, 13, and 14 of the estrous cycle from heifers with normal and high circulating progesterone. A total of 99 biochemicals (79 amino acids and 20 carbohydrates) were consistently identified, of which 31 showed a day by progesterone interaction. Fructose and mannitol/sorbitol did not exhibit a day by progesterone interaction, but displayed the greatest individual fluctuations (P ≤ 0.05) with respective fold increases of 18.39 and 28.53 in high vs normal progesterone heifers on day 12, and increases by 10.70-fold and 14.85-fold in the uterine fluid of normal progesterone animals on day 14 vs day 12. Moreover, enrichment analyses revealed that the phenylalanine, glutathione, polyamine, and arginine metabolic pathways were among the most affected by day and progesterone. In conclusion, progesterone had a largely stabilizing effect on amino acid flux, and identified biochemicals of likely importance to conceptus elongation initiation include arginine, fructose, glutamate, and mannitol/sorbitol.