Gray mold, caused by Botrytis cinerea is an intractable fungal disease that causes extensive damage to agricultural products. In the search for novel antifungal active ingredients, we discovered a linear pyranocoumarin Pd-D-V was effective against B. cinerea in both in vitro and in vivo assays. Furthermore, this study investigated the effects of Ca2+ and the Ca2+-calcineurin signaling pathway on its antifungal activity against B. cinerea. The results indicated that Pd-D-V reduced the concentration of Ca2+ in the mycelia of B. cinerea; CaCl2, the Ca2+ channel blocker verapamil, or the calcineurin inhibitor cyclosporin A could affect the sensitivity of Pd-D-V against B. cinerea; the expression of genes (Bccch1, Bcmid1, BccnA, Bccnb1, Bcpmc1, and Bcpmr1) of the Ca2+-calcineurin signaling pathway decreased after Pd-D-V treatment. In summary, Pd-D-V is compound for developing fungicides against B. cinerea. Pd-D-V can reduce intracellular Ca2+ concentration and disturb Ca2+ homeostasis. The Ca2+-calcineurin signaling pathway is important in the antifungal activity of Pd-D-V against B. cinerea.