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Aims: To investigate the active ingredient in fruiting bodies and to produce it with cultured mycelium in Antrodia camphorata (BCRC 35398). Methods and Results: The volatile components from the fruiting bodies, the liquid cultured broth of A. camphorata and Cinnamomum kanehirae wood were separately isolated by steam distillation–solvent extraction and identified by gas chromatography–mass spectrometry. In the fruiting bodies, a COX-2 inhibitor 2,4,5-trimethoxybenzaldehyde (TMBA) was found to be the most abundant constituent, but was totally absent in its cultured broth and its natural host, C. kanehirae wood. On feeding with the acid-digested sawdust of C. kanehirae wood or vanillin to the broth for culture, TMBA was produced in both cultured broths. Conclusion: The TMBA identified in fruiting bodies was an active ingredient whose functions consisted with the reported experiences of this mushroom. Feeding vanillin to culture broth could produce TMBA containing mycelium product like its fruiting bodies did. Significance and Impact of the Study: This study found an active ingredient in fruiting bodies of A. camphorata and elucidated this compound derived from digested sawdust of C. kanehirae wood. A feasible method was also developed to produce TMBA containing mycelium by feeding vanillin. [ABSTRACT FROM AUTHOR]