Mitochondrial defects are emerging as key drivers of gut inflammation, colitis, and IBD. Yet, their role in regulating immune tolerance, fine-tuned by helper T cells, is unclear. Understanding how metabolic changes in T cells impact gut tolerogenic mechanisms, including gut colonization and the balance between tolerance and disease, remains, therefore, a critical challenge. Here, we show that cardiolipin deficiency impairs tolerance to Helicobacter due to loss of regulatory T (Treg) cell function. Without cardiolipin, Treg cells lose metabolic fitness and immunosuppressive capacity igniting inflammation, particularly in the gut. Hence, mice with a T cell-specific deletion of the enzyme PTPMT1, essential for cardiolipin synthesis, are highly vulnerable to pathobiont infections even without microbiome imbalance. Notably, patients affected by Barth syndrome, a genetic disease characterized by severe cardiolipin deficiency, also exhibit inflammation and helper T cell imbalance. Overall, we found that cardiolipin in T cells preserves gut-immune homeostasis, dictating outcomes in pathobiont infections.