We have investigated the effect of chronic competitive inhibition of SNAT2 (System A) amino acid (AA) transport, induced by incubation with a saturating dose of a non-metabolisable System A amino acid analogue (Me-AIB), on growth and proliferation of MCF-7 human breast cancer cells in complete culture medium. These cells express Na+- and pH-dependent SNAT2 AA transport and a saturating concentration of Me-AIB (10 mM) competitively inhibits (>90%) AA uptake via SNAT2. Incubation with Me-AIB for up to 5 days progressively reduced cell proliferation (~2-fold) and depleted intracellular concentrations of not only SNAT2 AA substrates but of essential branched chain AAs (e.g. leucine). Surprisingly, total cellular protein was maintained and cells subjected to chronic Me-AIB incubation exhibited a detectable increase in cell size. Analysis of mTOR signalling revealed that, despite a substantial reduction in size of the intracellular AA pool, Me-AIB elevated mTOR-dependent p70S6K1 phosphorylation. Proteomic analysis of TAP-tag purified SNAT2 fusion proteins identified two novel SNAT2-interacting proteins that may potentially function in conjunction with the SNAT2 transceptor to regulate signalling pathways influencing protein turnover and cell growth.