We previously elucidated the pleotropic role of solute carrier family A1 member 5 (SLC1A5) as the primary transporter of glutamine (Gln), a modulator of cell growth and oxidative stress in non‐small cell lung cancer (NSCLC). The aim of our study was to evaluate SLC1A5 as a potential new therapeutic target and candidate biomarker predictive of survival and response to therapy. SLC1A5 targeting was examined in a panel of NSCLC and human bronchial cell lines by RNA interference and by a small molecular inhibitor, gamma‐l‐glutamyl‐p‐nitroanilide (GPNA). The effects of targeting SLC1A5 on cell growth, Gln uptake, ATP level, autophagy and cell death were examined. Inactivation of SLC1A5 genetically or pharmacologically decreased Gln consumption, inhibited cell growth, induced autophagy and apoptosis in a subgroup of NSCLC cell lines that overexpress SLC1A5. Targeting SLC1A5 function decreased tumor growth in NSCLC xenografts. A multivariate Cox proportional hazards analysis indicates that patients with increased SLC1A5 mRNA expression have significantly shorter overall survival (p = 0.01, HR = 1.24, 95% CI: 1.05–1.46), adjusted for age, gender, smoking history and disease stage. In an immunohistochemistry study on 207 NSCLC patients, SLC1A5 protein expression remained highly significant prognostic value in both univariate (p < 0.0001, HR = 1.45, 95% CI: 1.15–1.50) and multivariate analyses (p = 0.04, HR = 1.22, 95% CI: 1.01–1.31). These results position SLC1A5 as a new candidate prognostic biomarker for selective targeting of Gln‐dependent NSCLC. What's New? New strategies to overcome lung cancer mortality depend heavily on the discovery of novel therapeutic targets. In non‐small cell lung cancer (NSCLC), a possible target is solute linked carrier family 1A, member 5 (SLC1A5), a major glutamine transporter in NSCLC. This study furthers the promise of SLC1A5 by showing that its expression levels in lung cancer cells can predict cell sensitivity to the inhibitor gamma‐L‐glutamyl‐p‐nitroanilide (GPNA). In NSCLC cell lines, SLC1A5 inactivation led to glutamine starvation and oxidative stress‐mediated autophagy and apoptosis. In NSCLC patients, SLC1A5 expression was associated with poor overall survival.