Melittin, a peptide from bee venom, is thought to be a phospholipase A₂ activator and Ca²⁺ influx inducer that can evoke cell death in different cell types. However, the effect of melittin on cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) and viability has not been explored in human osteoblast-like cells. This study examined whether melittin altered [Ca²⁺]_i and killed cells in MG63 human osteosarcoma cells. [Ca²⁺]_i changes and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Melittin at concentrations above 0.075 μM increased [Ca²⁺]_i in a concentration-dependent manner. The Ca²⁺ signal was abolished by removing extracellular Ca²⁺. Melittin-induced Ca²⁺ entry was confirmed by Mn²⁺ quenching of fura-2 fluorescence at 360 nm excitation wavelength which was Ca²⁺-insensitive. The melittin-induced Ca²⁺ influx was unchanged by modulation of protein kinase-C activity with phorbol 12-myristate 13-acetate (PMA) and GF 109203X, or inhibition of phospholipase A₂ with AACOCF₃ and aristolochic acid; but was substantially inhibited by blocking L-type Ca²⁺ channels. At concentrations of 0.5 μM and 1 μM, melittin killed 33% and 45% of cells, respectively, via inducing apoptosis. Lower concentrations of melittin failed to kill cells. The cytotoxic effect of 1 μM melittin was completely reversed by pre-chelating cytosolic Ca²⁺ with BAPTA. Taken together, these data showed that in MG63 cells, melittin induced a [Ca²⁺]_i increase by causing Ca²⁺ entry through L-type Ca²⁺ channels in a manner independent of protein kinase-C and phospholipase A₂ activity; and this [Ca²⁺]_i increase subsequently caused apoptosis.