Abstract Several recent proteomic studies of plant peroxisomes indicate that the peroxisomal matrix harbors multiple ATP-dependent enzymes and chaperones. However, it is unknown whether plant peroxisomes are able to produce ATP by substrate-level phosphorylation or whether external ATP fuels the energy-dependent reactions within peroxisomes. The existence of transport proteins that supply plant peroxisomes with energy for fatty acid oxidation and other ATP-dependent processes has not previously been demonstrated. Here, we describe two Arabidopsis thaliana genes that encode peroxisomal adenine nucleotide carriers, PNC1 and PNC2. Both proteins, when fused to enhanced yellow fluorescent protein, are targeted to peroxisomes. Complementation of a yeast mutant deficient in peroxisomal ATP import and in vitro transport assays using recombinant transporter proteins revealed that PNC1 and PNC2 catalyze the counterexchange of ATP with ADP or AMP. Transgenic Arabidopsis lines repressing both PNC genes were generated using ethanol-inducible RNA interference. A detailed analysis of these plants showed that an impaired peroxisomal ATP import inhibits fatty acid breakdown during early seedling growth and other β-oxidation reactions, such as auxin biosynthesis. We show conclusively that PNC1 and PNC2 are essential for supplying peroxisomes with ATP, indicating that no other ATP generating systems exist inside plant peroxisomes.