The transcription factor nuclear factor erythroid 2-like 1 (NFE2L1 or NRF1) is involved in various critical cell processes such as maintenance of ubiquitin-proteasome system and regulation of the cellular antioxidant response. We previously determined that pancreatic β-cell-specific Nfe2l1-knockout mice had hyperinsulinemia and that silencing of Nfe2l1 in mouse islets or MIN6 insulinoma β-cells induced elevated basal insulin release and altered glucose metabolism. Hypoglycemia is a major issue with aggressive insulinomas, although a role of NFE2L1 in this pathology is not defined. In the present work, we studied the tumorigenicity of Nfe2l1-deficient insulinoma MIN6 cells (Nfe2l1-KD) and sensitivity to chemotherapy. Nfe2l1-KD cells grew faster and were more aggressive than Scramble cells in vitro. In a mouse allograft transplantation model, insulinomas arising from Nfe2l1-KD cells were more aggressive and chemoresistant. The conclusion was amplified using streptozotocin (STZ) administration in an allograft transplantation model in diabetic Akita background mice. Furthermore, Nfe2l1-KD cells were resistant to damage by the chemotherapeutic drugs STZ and 5-fluorouracil, which was linked to binding of hexokinase 1 with mitochondria, enhanced mitochondrial membrane potential and closed mitochondrial potential transition pore. Overall, both in vitro and in vivo data from Nfe2l1-KD insulinoma cells provided evidence of a previously un-appreciated action of NFE2L1 in suppression of tumorigenesis. Nfe2l1 silencing desensitizes insulinoma cells and derived tumors to chemotherapeutic-induced damage, likely via metabolic reprograming. These data indicate that NFE2L1 could potentially play an important role in the carcinogenic process and impact chemosensitivity, at least within a subset of pancreatic endocrine tumors.