background: Botrytis cinerea causes grey mould and is one of the most destructive fungal pathogens affecting important fruit and vegetable crops. In preliminary studies, we found that disenecioyl-cis-khellactone (DK) had strong antifungal activity against several fungi species including B. cinerea [half maximal effective concentration (EC )?=?11.0?μg?mL ]. In this study, we aimed to further evaluate the antifungal activity of DK against B. cinerea and determine the role of calcium ion/calcineurin (Ca /CN) signalling pathway on its antifungal effect. results: DK was effective against B. cinerea in both in vitro and in vivo assays. Exogenous Ca reduced the antifungal activity of DK. The combination of DK and cyclosporine A (CsA) did not exhibit an additive effect against B. cinerea. In contrast to CsA, DK reduced the intracellular Ca concentration in B. cinerea. DK bound to calcineurin A (cnA) and up-regulated the expression of PMC1 and PMR1 genes. Moreover, DK sensitivity of △bccnA significantly decreased compared with that of Bc05.10 strain. conclusions: DK is a promising lead compound for developing fungicides against B. cinerea. The Ca /CN signalling pathway plays a crucial role in the DK antifungal activity, and cnA is one of the targets of DK against B. cinerea. DK directly reacts with cnA, which up-regulates the transcription of Ca /CN-dependent target genes PMC1 and PMR1, decreasing the intracellular Ca concentration and disturbing the intracellular Ca balance, leading to cell death. ? 2022 Society of Chemical Industry.