AMPK and mTORC1 are nutrient-sensitive protein kinases that form a fundamental negative feedback loop governing cell growth and proliferation. AMPK is an αβγ heterotrimer expressing multiple isoforms of each subunit (α1/2, β1/2, γ1/2/3), whereby α2-S345 was shown to be directly phosphorylated by mTORC1 to suppress its activity. Using mass spectrometry, we generated precise phosphorylation profiles of all 12 AMPK complexes expressed in proliferating human cells. Of the 18 phosphorylation sites detected, several are previously uncharacterised and sensitive to pharmacological mTORC1 inhibition, including four in the unique NH2-terminal extension of the γ2 isoform, α2-S377, which is located in the AMPK nucleotide-sensing motif, and the conserved β1-S182 and β2-S184 (β-S182/4) residues. β-S182/4 in particular is the most heavily phosphorylated under cellular growth conditions and was identified as a direct mTORC1 substrate in vitro. β-S182/4 phosphorylation was elevated in α1-containing AMPK complexes relative to α2, and this effect was partly attributable to the poorly conserved α-subunit serine/threonine-rich loop, a target of growth factor signalling in tumour cells. While mutation of β-S182/4 to a non-phosphorylatable Ala residue had no effect on basal and ligand-stimulated AMPK activity, the β2-S184A mutation, but not its β1-S182A counterpart, preferentially increased nuclear AMPK activity to enhance cell proliferation in response to nutrient stress. Our findings demonstrate that mTORC1 governs nuclear AMPK activity and may reconcile, in part, the complex role AMPK plays in cancer whereby in the established tumour, nuclear AMPK endows cancerous cells with an ability to proliferate in a nutrient-scant environment.