21Targeting glycolysis has been considered therapeutically intractable owing to its essential 22 housekeeping role. However, the context-dependent requirement for individual glycolytic steps 23 has not been fully explored. We show that CRISPR-mediated targeting of glycolysis in T cells in 24 mice results in global loss of Th17 cells, whereas deficiency of the glycolytic enzyme glucose 25 phosphate isomerase (Gpi1) selectively eliminates inflammatory encephalitogenic and 26 colitogenic Th17 cells, without substantially affecting homeostatic microbiota-specific Th17 27 cells. In homeostatic Th17 cells, partial blockade of glycolysis upon Gpi1 inactivation was 28 compensated by pentose phosphate pathway flux and increased mitochondrial respiration. In 29 contrast, inflammatory Th17 cells experience a hypoxic microenvironment known to limit 30 mitochondrial respiration, which is incompatible with loss of Gpi1. Our study suggests that 31 inhibiting glycolysis by targeting Gpi1 could be an effective therapeutic strategy with minimum 32 toxicity for Th17-mediated autoimmune diseases, and, more generally, that metabolic 33 redundancies can be exploited for selective targeting of disease processes. 34 35 36 37 38 3 Introduction 39 40Cellular metabolism is a dynamic process that supports all aspects of the cell's activities. It 41