Background: Kidney Disease of unknown etiology (CKDu) in Sri Lanka is not related to the diabetes-related kidney damage but the extensive contamination of groundwater by fluoride was one of the main suspected reasons for the crises. Therefore, the aim of this study was to determine the effect of fluoride on kidney cell viability with exposure to increasing concentrations of fluoride for different duration of time periods. Method: Kidneys from four weeks old mice were removed aseptically and cultured with α-MEM media supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin / Streptomycin solution, overnight incubation at 370 C in a CO2 incubator. After 2 days, cells were divided into 0.5 ? 106 cells / 25cm2 culture flasks and 1x104 cells/ well in 96-well plates. Culture flasks were treated with Sodium fluoride (NaF) at concentrations of 0.125, 0.25, 0.5, 1, 2, 4 and 8 mM for 12 hr and the viable cell number was calculated by trypan blue assay. Then 96-well plates were exposed to same concentrations of NaF for 12 - 36 hr and mitochondrial activity was evaluated by using 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyl tetrazolium bromide (MTT) assay. Results: Renal cell damage was significantly decreased at 1, 2, 4 and 8mM NaF concentrations compared to control group with 20.13%, 28.5%, 30.88% and 38.45% respectively. The mitochondrial activity of renal cells showed a significant decrease at 8Mm NaF concentration after 12h. Moreover, by increasing the incubation time from 12 to 24h and 36h, the mitochondrial activity was further decreased at 2, 4 and 8mM NaF at 24h and 0.5, 1, 2, 4 and 8Mm NaF at 36h with respect to control groups. Conclusion: Fluoride exposure resulted renal cell damage and concentration dependent decrease in mitochondrial activity with increased exposure time.