Pancreatic cancer exhibits pronounced glutamine dependency, but monotherapeutic use of the glutaminase inhibitor CB-839 faces limitations due to adaptive metabolic reprogramming. This study demonstrates that while CB-839 suppresses tumor cell proliferation, it concurrently activates the GCN2-ATF4 signaling axis. ATF4 upregulates the glutamine transporter ASCT2, facilitating nutrient uptake and stress survival. Combination treatment with CB-839 and the ASCT2 inhibitor V-9302 disrupts this compensatory mechanism, inducing apoptosis. The dual targeting strategy synergistically reduces pancreatic cancer cell viability, underscoring GLS1 and ASCT2 as pivotal therapeutic targets. These results suggest co-inhibition of GLS and ASCT2 to counteract metabolic plasticity and improve clinical outcomes.