Secondary metabolites derived from benzoic acid (BA) are of central importance in the interactions of plants with pests, pathogens and symbionts, and potentially important in plant development. Peroxisomal β-oxidation has recently been shown to contribute to BA biosynthesis in plants, but not all of the enzymes involved have been defined. In this report, we demonstrate that the peroxisomal ABC transporter COMATOSE (CTS) is required for accumulation of benzoylated secondary metabolites in Arabidopsis seeds, including benzoylated glucosinolates and substituted hydroxybenzoylcholines. The ABNORMAL INFLORESCENCE MERISTEM (AIM1) protein, one of two multifunctional proteins encoded by Arabidopsis, is essential for accumulation of these compounds, and MFP2 contributes to synthesis of substituted hydroxybenzoylcholines. Of the two major 3-ketoacyl CoA thiolases, KAT2 plays the primary role in BA synthesis. Thus BA biosynthesis in Arabidopsis employs the same core set of β-oxidation enzymes as in the synthesis of indole-3-acetic acid from indole-3-butyric acid.