Molecular targets for breast cancer therapy are highly sought after, particularly in an aggressive subgroup known as triple-negative breast cancer (TNBC) which is insensitive to hormone therapies. As many breast cancers display dependence on the amino acid glutamine, the glutamine transporter ASCT2 is considered a promising target [1]. However, the activation of alternative nutrient uptake mechanisms such as macropinocytosis, a mechanism of non-specific macromolecule uptake, may cause resistance to therapies targeting nutrient transporters [2]. We aimed to observe the effects of ASCT2 inhibition in breast cancer, and to determine whether macropinocytosis is activated in TNBC following transporter loss. CRISPR-Cas9 ASCT2 knockouts (KO) were generated from six breast cancer cell lines. Amino acid uptake and cell viability in each line was examined and compared to matched controls. RNA sequencing (RNA-Seq) analysis was performed on the glutamine-addicted TNBC cell line HCC1806 (control and KO) and compared to HCC1806 ASCT2 shRNA knockdown (KD) lines. To quantify macropinocytosis in HCC1806 lines, uptake of high-molecular-weight dextran molecules was measured by fluorescence microscopy. In HCC1806 cells, ASCT2 KD markedly inhibited glutamine uptake and cell growth, while cell growth following KO was unaffected. Following KO in five additional breast cancer cell lines, growth was again unaffected in all but one. We proposed that compensatory mechanisms may be activated following KO but not KD. Indeed, HCC1806 ASCT2 KO lines displayed a significant induction of macropinocytosis. Through RNA-Seq, potential drivers of this macropinocytic phenotype were identified – a sorting nexin protein known as SNX33, and a roundabout receptor known as ROBO1. In summary, contrary to the results of previous RNA interference studies, TNBC and other breast cancer lines are unaffected by loss of ASCT2. This may be attributed to a compensatory elevation of macropinocytosis, and prompts caution when considering this transporter, or nutrient transporters more broadly, as molecular targets.