Pancreatic ductal adenocarcinoma (PDA) cells reprogram their transcriptional and metabolic programs to survive the nutrient-poor tumor microenvironment. Through in vivo CRISPR screening, we discovered islet-2 (ISL2) as a candidate tumor suppressor that modulates aggressive PDA growth. Notably, ISL2, a nuclear and chromatin-associated transcription factor, is epigenetically silenced in PDA tumors and high promoter DNA methylation or its reduced expression correlates with poor patient survival. The exogenous ISL2 expression or CRISPR-mediated upregulation of the endogenous loci reduces cell proliferation. Mechanistically, ISL2 regulates the expression of metabolic genes, and its depletion increases oxidative phosphorylation (OXPHOS). As such, ISL2-depleted human PDA cells are sensitive to the inhibitors of mitochondrial complex I in vitro and in vivo. Spatial transcriptomic analysis shows heterogeneous intratumoral ISL2 expression, which correlates with the expression of critical metabolic genes. These findings nominate ISL2 as a putative tumor suppressor whose inactivation leads to increased mitochondrial metabolism that may be exploitable therapeutically. [Display omitted] • In vivo CRISPR screening identified ISL2 as a tumor suppressor • ISL2 is epigenetically silenced in a large fraction of pancreatic tumors • ISL2 downregulation reprograms the metabolism of pancreatic cancer cells • ISL2 inactivation can be exploitable therapeutically Ozturk et al. identify ISL2 as a tumor suppressor of pancreatic cancer growth. They find that ISL2 is epigenetically silenced in a significant fraction of pancreatic tumors, and reduced ISL2 expression reprograms metabolic genes in cancer cells to rely on mitochondrial metabolism rather than glycolysis. [ABSTRACT FROM AUTHOR]