ABSTRACT:: The effect of the natural product 3,3′-diindolylmethane (DIM) on cytosolic Ca concentrations ([Ca]i) and viability in MG63 human osteosarcoma cells was explored. The Ca-sensitive fluorescent dye fura-2 was applied to measure [Ca]i. DIM at concentrations of 40–80 μM induced a [Ca]i rise in a concentration-dependent manner. The response was reduced partly by removing Ca. DIM-evoked Ca entry was suppressed by nifedipine, econazole, SK&F96365 and protein kinase C modulators. In the absence of extracellular Ca, incubation with the endoplasmic reticulum Ca pump inhibitors thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited or abolished DIM-induced [Ca]i rise. Incubation with DIM also inhibited thapsigargin or BHQ-induced [Ca]i rise. Inhibition of phospholipase C with U73122 abolished DIM-induced [Ca]i rise. At concentrations of 10–50 μM, DIM killed cells in a concentration-dependent manner. This cytotoxic effect was not altered by chelating cytosolic Ca with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). Annexin V/propidium iodide staining data implicate that DIM (20 and 40 μM) induced apoptosis in a concentration-dependent manner. In sum, in MG63 cells, DIM induced a [Ca]i rise by evoking phospholipase C-dependent Ca release from the endoplasmic reticulum and Ca entry via protein kinase C-sensitive store-operated Ca channels. DIM caused cell death that may involve apoptosis.