Diffusion barriers enable plant survival under fluctuating environmental conditions. They control internal water potential and protect against biotic or abiotic stress factors. How these protective molecules are deposited to the extracellular environment is poorly understood. We here examined the role of the Arabidopsis ABC half-size transporter AtABCG1 in the formation of the extracellular root suberin layer. Quantitative analysis of extracellular long-chain fatty acids and aliphatic alcohols in the atabcg1 mutants demonstrated altered root suberin composition, specifically a reduction in longer chain dicarboxylic acids, fatty alcohols and acids. Accordingly, the ATP-hydrolyzing activity of heterologous expressed and purified AtABCG1 was strongly stimulated by fatty alcohols (C26-C30) and fatty acids (C24-C30) in a chain length dependent manner. These results are a first indication for the function of AtABCG1 in the transport of longer chain aliphatic monomers from the cytoplasm to the apoplastic space during root suberin formation.