Recently, we reported that baicalein 5,6,7-trimethyl ether (BTM), a flavonoid, is capable of activating fatty acid β-oxidation in X-linked adrenoleukodystrophy (X-ALD) fibroblasts (FEBS Lett. 2005; 579: 409–414). The objective of this study was to clarify whether BTM activates peroxisomal and/or mitochondrial fatty acid β-oxidation. We first analysed the effect of BTM on fatty acid β-oxidation in fibroblasts derived from healthy controls as well as patients with X-ALD, mitochondrial carnitine-acylcarnitine translocase (CACT) deficiency, and peroxisome biogenesis disorder, Zellweger syndrome. Lignoceric acid (C24:0) β-oxidation in the fibroblasts was stimulated by treatment with BTM, except for Zellweger fibroblasts. In contrasts, palmitic acid (C16:0) β-oxidation was increased (2.8-fold) only in CACT-deficient fibroblasts. In U87 glioblastoma cells, C24:0 β-oxidation was also activated by treatment with BTM but C16:0 β-oxidation was not. The C16:0 β-oxidation was, however, significantly increased in the presence of 2-[5-(4-chlorophenyl)pentyl]oxirane-2-carboxylate (POCA), a carnitine palmitoyltransferase I inhibitor. These results indicate that BTM activates peroxisomal but not mitochondrial fatty acid β-oxidation. In addition, we found that BTM did not upregulate the expression of ABCD2/ALDR, ABCD3/PMP70, ACOX1 and FATP4 genes but slightly increased ACSVL1 gene expression.