Description: Dysregulation of receptor tyrosine kinases (RTK) contributes to cellular transformation and cancer progression by disrupting key metabolic signaling pathways. The EPHA2 RTK is overexpressed in aggressive forms of breast cancer, including the HER2+ subtype, and correlates with poor prognosis. However, the role of EPHA2 in tumor metabolism remains unexplored. In this study, we used in vivo and in vitro models of HER2-overexpressing breast cancer to investigate the mechanisms by which EPHA2 ligand–independent signaling promotes tumorigenesis in the absence of its prototypic ligand, ephrin-A1. We demonstrate that ephrin-A1 loss leads to upregulated glutamine metabolism and lipid accumulation that enhanced tumor growth. Global metabolic profiling of ephrin-A1–null, HER2-overexpressing mammary tumors revealed a significant increase in glutaminolysis, a critical metabolic pathway that generates intermediates for lipogenesis. Pharmacologic inhibition of glutaminase activity reduced tumor growth in both ephrin-A1–depleted and EPHA2-overexpressing tumor allografts in vivo. Mechanistically, we show that the enhanced proliferation and glutaminolysis in the absence of ephrin-A1 were attributed to increased RhoA-dependent glutaminase activity. EPHA2 depletion or pharmacologic inhibition of Rho, glutaminase, or fatty acid synthase abrogated the increased lipid content and proliferative effects of ephrin-A1 knockdown. Together, these findings highlight a novel, unsuspected connection between the EPHA2/ephrin-A1 signaling axis and tumor metabolism, and suggest potential new therapeutic targets in cancer subtypes exhibiting glutamine dependency. Cancer Res; 76(7); 1825–36. ©2016 AACR.