During the peri-implantation and early placentation periods in pigs, conceptuses (embryo and its extra-embryonic membranes) undergo dramatic morphological changes and differentiation that require the exchange of nutrients (histotroph) and gasses across the trophectoderm and a true epitheliochorial placenta. Of these nutrients, arginine (Arg), leucine (Leu), and glutamine (Gln) are essential components of histotroph; however, little is known about changes in their total amounts in the uterine lumen of cyclic and pregnant gilts and their effects on cell signaling cascades. Therefore, we determined quantities of Arg, Leu, and Gln in uterine luminal fluids and found that total recoverable amounts of these amino acids increased in pregnant but not cyclic gilts between Days 12 and 15 after onset of estrus. We hypothesized that Arg, Leu, and Gln have differential effects on hypertrophy, hyperplasia, and differentiated functions of trophectoderm cells that are critical to conceptus development. Primary porcine trophectoderm (pTr) cells treated with either Arg, Leu, or Gln had increased abundance of phosphorylated RPS6K, RPS6, and EIF4EBP1 compared to basal levels, and this effect was maintained for up to 120 min. When pTr cells were treated with Arg, Leu, and Gln, low levels of pRPS6K and pEIF4EBP1 were detected in the cytosol, but the abundance of nuclear pRPS6K increased. Immunofluorescence analyses revealed abundant amounts of pRPS6 protein in the cytoplasm of pTr cells treated with Arg, Leu, and Gln. These amino acids also increased proliferation of pTr cells. Furthermore, when Arg, Leu, and Gln were combined with siRNAs for either MTOR, RPTOR, or RICTOR, effects of those amino acids on proliferation of pTr cells were significantly inhibited. Collectively, these results indicate that Arg, Leu, and Gln act coordinately to stimulate proliferation of pTr cells through activation of the MTOR-RPS6K-RPS6-EIF4EBP1 signal transduction pathway.