In higher plants, formate dehydrogenase (FDH; EC 1.2.1.2) is a NAD-dependent enzyme that catalyzes the oxidation of formate into carbon dioxide. Although FDHs have been reported to be induced by various abiotic stresses, the function of FDHs in biotic stress is rarely known. In this work, we describe the identification of pepper mitochondrial FORMATE DEHYDROGENASE1 (CaFDH1) as a positive regulator of cell death and disease resistance. Transient expression of CaFDH1 in Nicotiana benthamiana leaves caused hypersensitive response (HR)-like cell death, where D-isomer specific 2-hydroxyacid dehydrogenase signature was crucial for the induction of the cell death. Localization analysis using green fluorescence protein showed that CaFDH1 targeted to the mitochondria with the mitochondria targeting sequence at N-terminal region. However, the mitochondrial localization of CaFDH1 is not essential for the induction of HR-like cell death, because the cytosolic CaFDH1 could elicit cell death response. Silencing of CaFDH1 in pepper significantly compromised the cell death response and salicylic acid (SA) levels, but enhanced growth of Xanthomonas campestris pv. vesicatoria. In contrast, transgenic Arabidopsis overexpressing FDH1 exhibited enhanced resistance to Pseudomonas syringae pv. tomato. Taken together, these data suggest that CaFDH1 has a role in HR cell death and defense response to microbial pathogens.