The LuPLR1 gene encodes a pinoresinol lariciresinol reductase responsible for the biosynthesis of (+)-secoisolariciresinol, a cancer chemopreventive lignan, highly accumulated in the seedcoat of flax (Linum usitatissimum L.). Abscisic acid (ABA) plays a key role in the regulation of LuPLR1 gene expression and lignan accumulation in both seeds and cell suspensions, which require two cis-acting elements (ABRE and MYB2) for this regulation. Ca2+ is a universal secondary messenger involved in a wide range of physiological processes including ABA signaling. Therefore, Ca2+ may be involved as a mediator of LuPLR1 gene expression and lignan biosynthesis regulation exerted by ABA. To test the potential implication of Ca2+ signaling, a pharmacological approach was conducted using both flax cell suspensions and maturing seed systems coupled with a ß-glucuronidase reporter gene experiment, RT-qPCR analysis, lignan quantification as well as Ca2+ fluorescence imaging. Exogenous ABA application results in an increase in the intracellular Ca2+ cytosolic concentration, originating mainly from the extracellular medium. Promoter-reporter deletion experiments suggest that the ABRE and MYB2 cis-acting elements of the LuPLR1 gene promoter functioned as Ca2+-sensitive sequences involved in the ABA-mediated regulation. The use of specific inhibitors pointed the crucial roles of the Ca2+ sensors calmodulin-like proteins and Ca2+-dependent protein kinases in this regulation. This regulation appeared conserved in the two different studied systems, i.e. cell suspensions and maturing seeds. A calmodulin-like, LuCML15b, identified from gene network analysis is proposed as a key player involved in this signal transduction since RNAi experiments provided direct evidences of this role. Taken together, these results provide new information on the regulation of plant defense and human health-promoting compounds, which could be used to optimize their production.