The mammalian target of rapamycin (mTOR) pathway is activated by a variety of stimuli, including nutrients such as glucose and amino acids. The Ste20 family kinase MAP4K3 is regulated by amino acids and acts upstream of mTORC1. Here we investigate how MAP4K3 activity is regulated by amino acid sufficiency. We identify a transautophosphorylation site in the MAP4K3 kinase activation segment (Ser170) that is required for MAP4K3 activity and its activation of mTORC1 signaling. Following amino acid withdrawal, Ser170 is dephosphorylated via PP2A complexed to PR61e, a PP2A-targeting subunit. Inhibition of PR61e expression prevents MAP4K3 Ser170 dephosphorylation and impairs mTORC1 inhibition during amino acid withdrawal. We propose that during amino acid sufficiency Ser170-phosphorylated MAP4K3 activates mTORC1, but that upon amino acid restriction MAP4K3 preferentially interacts with PP2AT61e, promoting dephosphorylation of Ser170, MAP4K3 inhibition, and, subsequently, inhibition of mTORC1 signaling.