Reversible post-translational modifications represent a mechanism to control tumor metabolism. Here we show that mitochondrial Sirtuin5 (SIRT5), which mediates lysine desuccinylation, deglutarylation, and demalonylation, plays a role in colorectal cancer (CRC) glutamine metabolic rewiring. Metabolic profiling identifies that deletion of SIRT5 causes a marked decrease in C-glutamine incorporation into tricarboxylic-acid (TCA) cycle intermediates and glutamine-derived non-essential amino acids. This reduces the building blocks required for rapid growth. Mechanistically, the direct interaction between SIRT5 and glutamate dehydrogenase 1 (GLUD1) causes deglutarylation and functional activation of GLUD1, a critical regulator of cellular glutaminolysis. Consistently, GLUD1 knockdown diminishes SIRT5-induced proliferation, both in vivo and in vitro. Clinically, overexpression of SIRT5 is significantly correlated with poor prognosis in CRC. Thus, SIRT5 supports the anaplerotic entry of glutamine into the TCA cycle in malignant phenotypes of CRC via activating GLUD1.