In the present study, the effect of fluoride on intracellular free calcium ([Ca2+]i) and Ca2+-ATPase of renal cells were examined. Some paradoxical experimental results about the mechanism of fluoride toxicity were observed. In vivo, 48 Wistar rats were divided into 4 groups, and half of rats were treated with sodium fluoride (NaF) by drinking water (per liter of tap water containing 100 mg F-). Compared with the respective control, the level of [Ca2+]i of the kidney in two fluoride-treated rats obviously increased (p < 0.05); and the activity of Ca2+-ATPase in 100 mg F-/L groups with a standard diet did not significantly increase, and the enzyme activity in 100-mg F-/L group with a low-calcium diet decreased significantly compared to the 100 mg F-/L group with a standard diet (p < 0.05). In vitro, renal tubular cells were cultured and respectively exposed to 1.0, 5.0, 7.5, and 12.5 mg/L fluoride in the culture medium. Results showed the significantly elevated activity of Ca2+-ATPase in the cells exposed to 1.0 and 5.0 mg/L fluoride (p < 0.05), and this enzyme activity indicated inhibitory trend in cells of the 7.5- and 12.5-mg/L fluoride-treated group. To sum up, the effect of fluoride on Ca2+-ATPase is a similar to a dose-effect relationship phenomenon characterized by low-dose stimulation and high-dose inhibition, and the increase of [Ca2+]i probably plays a key role on the mechanism of renal injury in fluorosis.