In this study we report the molecular genetic characterization of the Arabidopsis mitochondrial phosphopantetheinyl transferase (mtPPT)， which catalyzes the phosphopantetheinylation and thus activation of mitochondrial acyl carrier protein (mtACP) of mitochondrial fatty acid synthase (mtFAS). This catalytic capability of the purified mtPPT protein (encoded by AT3G11470) was directly demonstrated in an in vitro assay that phosphopantetheinylated mature Arabidopsis apo-mtACP isoforms. The mitochondrial localization of the AT3G11470-encoded proteins was validated by the ability of their N-terminal 80-residue leader sequence to guide a chimeric GFP protein to this organelle. A T-DNA-tagged null mutant mtppt-1 allele shows an embryo-lethal phenotype， illustrating a crucial role of mtPPT for embryogenesis. Arabidopsis RNAi transgenic lines with reduced mtPPT expression display typical phenotypes associated with a deficiency in the mtFAS system， namely miniaturized plant morphology， slow growth， reduced lipoylation of mitochondrial proteins， and the hyperaccumulation of photorespiratory intermediates， glycine and glycolate. These morphological and metabolic alterations are reversed when these plants are grown in a non-photorespiratory condition (i.e. 1% CO2 atmosphere)， demonstrating that they are a consequence of a deficiency in photorespiration due to the reduced lipoylation of the photorespiratory glycine decarboxylase.