Very long chain fatty acid (VLCFA) metabolism in astrocytes is important for the maintenance of myelin structure in central nervous system. To analyze the contribution of the ABCD1-dependent and -independent pathways to VLCFA metabolism in astrocytes, we prepared human glioblastoma U87 cells with a silencing of ABCD1 and primary astrocytes from abcd1-deficient mice, and measured fatty acid β-oxidation in the presence or absence of a potent inhibitor of carnitine palmitoyltransferase I, 2-[5-(4-chlorophenyl)pentyl]oxirane-2-carboxylate (POCA). In U87 cells, C24:0 β-oxidation was decreased to ca. 70% of the control in the presence of POCA, and the activity was further decreased to ca. 20% by the silencing of ABCD1. In mouse primary astrocytes, C24:0 β-oxidation was also decreased to ca. 70% of the control in the presence of POCA. The C24:0 β-oxidation in Abcd1-deficient primary astrocytes was ca. 60% of the wild-type cells and the activity was further decreased to ca. 25% in the presence of POCA. Compared to human skin fibroblasts, in which VLCFA β-oxidation is not significantly inhibited by POCA, approximately one-third of the overall VLCFA β-oxidation was inhibited in both types of astrocytic cells. These results suggest that VLCFA is indeed β-oxidized in ABCD1-dependent pathway, but the ABCD1-independent peroxisomal and mitochondrial β-oxidation pathways significantly contribute to VLCFA β-oxidation in astrocytic cells.