Background: Urginea maritima (L.f.) Baker (Hyacinthaceae) is a perennial bulbous medicinal plant that is currently at risk of extinction. Squill (white sea onion) is an analogous cardiotonic to digitalis. The purpose of the current work was to assess the optimal growth conditions for Urginea cells to synthesize the cardiac glycoside proscillaridin A by involving illumination, carbon source, methyl jasmonate (MJ), and culture system. Results: When cells were cultured for 28 days at 21 \pm 2 {}^{\circ }C in the dark on Murashige and Skoog (MS) media containing 1 mg/L 2, 4-D and 0.5 mg/L Kin and 30 g/L sucrose, the cell proliferation, and proscillaridin A synthesis were effectively controlled. At low concentrations, MJ stimulated the synthesis of proscillaridin A (PsA). High-performance liquid chromatography (HPLC) analysis of suspension extracts demonstrated that the callus maintained in MS media enriched with 1 mg/L 2, 4-D and 0.5 mg/L Kin yielded a greater formation of Proscillaridin A (141.31 mg/g DW) than untreated plants. Conclusions: The results indicate that in vitro cultures of U. maritima may be an excellent source of proscillaridin A. Moreover, it is one of the most important cardiac glycoside, which has been found to exhibit anticancer activities. Suspension cultures of Urginea cells could be as highly productive as a callus culture.