The activity of placental amino acid transporters is decreased in intrauterine growth restriction (IUGR), but the underlying regulatory mechanisms are not established. Inhibition of the mammalian target of rapamycin (mTOR) signaling pathway has been shown to decrease the activity of the system L amino acid transporter in human placental villous fragments and placental mTOR activity is decreased in IUGR. In the current study we used cultured primary trophoblast cells to study mTOR regulation of placental amino acid transporters in more detail and to test the hypothesis that mTOR alters amino acid transport activity by changes in transporter expression. Inhibition of mTOR by rapamycin significantly reduced the activity of the system A (-17%), system L (-28%), and taurine (-40%) amino acid transporters. The mRNA expression of isoforms of the three amino acid transporter systems in response to mTOR inhibition was measured using RT-QPCR. LAT1, a system L isoform, and taurine transporter mRNA expression was reduced by 13% and 50% respectively, however mTOR inhibition did not alter the mRNA expression of system A isoforms (SNAT1, 2, and 4), LAT2 or 4F2hc. Rapamycin treatment did not significantly affect the protein expression of any of the transporter isoforms. We conclude that mTOR signaling regulates the activity of key placental amino acid transporters and that this effect is not due to a decrease in total protein expression. These data suggest that mTOR regulates placental amino acid transporters by posttranslational modifications or by affecting transporter translocation to the plasma membrane.