The ABCD1 protein, one of the four ATP-binding cassette (ABC) proteins in subfamily D, is located on the peroxisomal membrane and is involved in the transport of very long chain fatty acid (VLCFA)-CoA into peroxisomes. Its mutation causes X-linked adrenoleukodystophy (X-ALD): an inborn error of peroxisomal β-oxidation of VLCFA. Whether ABCD1 transports VLCFA-CoA as a CoA ester or free fatty acid is controversial. Recently, Comatose (CTS), a plant homologue of human ABCD1, has been shown to possess acyl-CoA thioesterase (ACOT) activity, and it is suggested that this activity is required for transport of acyl-CoA into peroxisomes. However, the precise transport mechanism is unknown. Here, we expressed human His-tagged ABCD1 in methylotrophic yeast, and characterized its ACOT activity and transport mechanism. The expressed ABCD1 possessed both ATPase and ACOT activities. The ACOT activity of ABCD1 was inhibited by p-chloromercuribenzoic acid (pCMB), a cysteine-reactive compound. Furthermore, we performed a transport assay with ABCD1-containing liposomes using 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD)-labeled acyl-CoA as the substrate. The results showed that the fatty acid produced from VLCFA-CoA by ABCD1 is transported into liposomes and that ACOT activity is essential during this transport process. We propose a detailed mechanism of VLCFA-CoA transport by ABCD1.