Dalesconols (dalesconols A and B) were isolated from Daldinia eschscholzii and have remarkable immunosuppressive activity. In this study, the response of fungal growth, intra- and extracellular Ca2+, and dalesconols production after CaCl2 addition were reported for the first time. After supplementation with 5 mM Ca2+ at 24 h, dalesconols production reached 84.33 mg/L, which resulted in a 1.57-fold enhancement compared to the control. The key role of calcium/calmodulin signaling in dalesconols biosynthesis was confirmed by treatment with Ca2+ channel and calmodulin inhibitors. The transcriptional levels of dalesconols biosynthetic genes were up-regulated after CaCl2 addition and down regulated after inhibitors were added. The results demonstrated that Ca2+ addition induces dalesconols biosynthesis through up-regulation of dalesconols biosynthesis genes via regulation of calcium/calmodulin signaling. This study provided an efficient strategy for improving dalesconols production and would facilitate further research on the biosynthesis and regulation of dalesconols. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.