SummaryThe aim was to determine the function of peroxisomal NAD+‐malate dehydrogenase (PMDH) in fatty acid β‐oxidation and the glyoxylate cycle in Arabidopsis. Seeds in which both PMDH genes are disrupted by T‐DNA insertions germinate, but seedling establishment is dependent on exogenous sugar. Mutant seedlings mobilize their triacylglycerol very slowly and growth is insensitive to 2,4‐dichlorophenoxybutyric acid. Thus mutant seedlings are severely impaired in β‐oxidation, even though microarray analysis shows that β‐oxidation genes are expressed normally. The mutant phenotype was complemented by expression of a cDNA encoding PMDH with either its native peroxisome targeting signal‐2 (PTS2) targeting sequence or a heterologous PTS1 sequence. In contrast to the block in β‐oxidation in mutant seedlings, [14C]acetate is readily metabolized into sugars and organic acids, thereby demonstrating normal activity of the glyoxylate cycle. We conclude that PMDH serves to reoxidize NADH produced from fatty acid β‐oxidation and does not participate directly in the glyoxylate cycle.