Lithium is the most commonly used drug for the treatment of manic-depressive illness. The precise mechanisms underlying its clinical efficacy remain unknown. In this study, we found that long-term exposure to lithium chloride protected cultured cerebellar granule neurons (CGNs) against β-bungarotoxin (β-BuTX)-induced neurotoxicity. This neuroprotection was exhibited at the therapeutically relevant concentration of 1.2 mM lithium. Pretreatments for 3–5 days (long-term) were required for protection to occur; but a 3 hr treatment (short-term) was ineffective. In contrast, a longer treatment for 6–7 days or a higher concentration of 3 mM lithium led not only to loss of the neuroprotective effect but also to a neurotoxic effect. These findings suggest that lithium protection is limited to its narrow window of concentration and apparently relevant to its narrow therapeutic index in clinical application. Measurement of intracellular calcium [Ca2+]i revealed that neurotoxic concentrations of β-BuTX markedly increased [Ca2+]i, which could be attenuated by long-term, but not short-term, lithium treatment. Thus, the protection induced by lithium in CGNs was attributed to its inhibition of calcium overload. In addition, the Ca2+ signaling pathway, including reactive oxygen species production and mitochondrial membrane potential reduction, along with the neurotoxic effect of β-BuTX was blocked by long-term, but not short-term, lithium treatment. All of these results indicate that a crucial step for lithium protection is modulation of [Ca2+]i homeostasis and that lithium neurotoxicity possibly, at least in part, is due to calcium overload. In conclusion, our results suggest that lithium, in addition to its use in treatment of bipolar depressive illness, may have an expanded use in intervention for neurotoxicity. © 2002 Wiley-Liss, Inc.