Maintaining mitochondrial proteome integrity is especially important under stress conditions to ensure a continued ATP supply for protection and adaptation responses in plants. Deg/HtrA proteases are important factors in the cellular protein quality control system, but little is known about their function in mitochondria. Here we analyzed the expression pattern and physiological function of Arabidopsis thaliana DEG10, which has homologs in all photosynthetic eukaryotes. Both expression of DEG10:GFP fusion proteins and immunoblotting after cell fractionation showed an unambiguous subcellular localization exclusively in mitochondria. DEG10 promoter:GUS fusion constructs showed that DEG10 is expressed in trichomes but also in the vascular tissue of roots and aboveground organs. DEG10 loss-of-function mutants were impaired in root elongation, especially at elevated temperature. Quantitative proteome analysis revealed concomitant changes in the abundance of mitochondrial respiratory chain components and assembly factors, which partially appeared to depend on altered mitochondrial retrograde signaling. Under field conditions, lack of DEG10 caused a decrease in seed production. Taken together, our findings demonstrate that DEG10 affects mitochondrial proteostasis, is required for optimal root development and seed set under challenging environmental conditions, and thus contributes to stress tolerance of plants.