Fluoride is known to induce apoptosis though the mechanisms remain obscure. The aim of the present study was to understand the underlying molecular mechanisms of fluoride-induced apoptosis using fish headkidney macrophages (HKMs). Exposure to fluoride triggered HKM cell apoptosis as evidenced by Hoechst 333432 and AnnexinV-propidium iodide staining, the presence of an internucleosomal DNA ladder and the comet assay. Our results suggest the influx of extra-cellular Ca2+ to be an initial event in fluoride-induced HKM cell apoptosis. We observed persistently elevated levels of superoxide anions and our inhibitor studies with EGTA suggested the primal role of the Ca2+ flux in triggering superoxide production in fluoride-exposed HKM cells. Fluoride exposure led to elevated levels of Ca2+/CaM dependent protein kinase II gamma (CaMKIIg) and pre-treatment with the inhibitor KN-93 but not its inactive structural analogue KN-92 reduced the number of apoptotic cells establishing the pro-apoptotic role of CaMKIIg in fluoride-induced HKM cell apoptosis. We report that the sustained superoxide generation is primarily responsible for the increased CaMKIIg levels observed in fluoride-exposed HKM cells. Our inhibitor studies further implicated CaMKIIg in the activation of extracellular signal-regulated kinases 1 and 2 (ERK 1/2) culminating in caspase-8/caspase-3 mediated apoptosis of HKM cells. We conclude that fluoride-induced apoptosis is largely dependent on Ca2+ induced superoxide generation leading to elevation in CaMKIIg which in turn induces the phosphorylation of ERK 1/2 and downstream activation of extrinsic caspase cascade in HKM cells.