KRAS^G13D colorectal cancer (CRC) exhibits distinct biological and therapeutic characteristics, suggesting genotype-specific vulnerabilities. Here, we investigated the role of HER2 in metabolic reprogramming of KRAS^G13D CRC. Untargeted and targeted metabolomic profiling revealed that HER2 depletion markedly altered amino acid metabolism, particularly glutamine–glutamate metabolism, accompanied by decreased intracellular glutamate and the glutamate-to-glutamine ratio. Transcriptomic and molecular analyses identified glutaminase 1 (GLS1) as a potential downstream metabolic effector of HER2, with preferential reduction of GLS1 following HER2 depletion in KRAS^G13D cells. Analysis of human CRC datasets further showed a positive association between ERBB2 and GLS expression and poor survival associated with high GLS expression in KRAS^G13D tumors. Functionally, KRAS^G13D cells were sensitive to glutamine deprivation and pharmacological or genetic GLS1 inhibition, while GLS1 inhibition enhanced the antitumor effect of cetuximab. GSEA further revealed enrichment of MYC target and PI3K–AKT signaling signatures in GLS-high KRAS^G13D tumors. Collectively, these findings identify a HER2-associated GLS1-dependent metabolic vulnerability in KRAS^G13D CRC and support GLS1 as a potential therapeutic target in this molecular subtype.