In addition to causing skeletal and dental fluorosis, fluoride (F) in drinking water may damage other organs including the thyroid. The objective of this study was to explore the toxicity of F on immortalized human normal thyroid cells (Nthy-ori 3-1) exposed to 0, 0.1, 1, and 3 mmol/L of sodium fluoride (NaF) in vitro. After 24 hours incubation, measurements were made of cell viability and of the lactate dehydrogenase (LDH) leakage rate by MTT and LDH assay. The reactive oxygen species (ROS) level, the constituent ratio of the cell cycle, and the apoptosis rate were measured by flow cytometry. Cell viability was decreased (p<0.01) in the 1 and 3 mmol/L F-treated groups (76.64±9.13% and 64.04±6.32% respectively) compared to the control group (100.00±0.00%). The LDH leakage rate and the ROS level were increased (p<0.01) in the 3 mmol/L F-treated group (48.66±7.15% and 29993.50±1786.86 FI respectively) compared to the control group (35.24±3.02%) and 13021.33±1067.55 FI). The % of G0/G1 phase cells was decreased (p<0.01) in the 1 mmol/L F-treated group (40.76±5.65%) compared to the control group (60.09±1.76)% but the % of S phase cells (54.05±4.59%) was increased (p<0.01) compared to the control group (32.59±2.43%). The % of apoptotic cells was increased (p<0.01) in the 3 mmol/L F-treated group (20.09±3.22%) compared to the control group (9.64±3.44%). The p53 mRNA expression was not changed in the F-treated groups (p>0.05) but in the 3 mmol/L F-treated groups the Bax mRNA expression increased (p<0.05) and the Bcl-2 mRNA expression decreased (p<0.05) compared to the control group. Thus F in Nthy-ori 3-1 cells may decrease cell viability, increase the LDH leakage rate and the ROS level, block growth in the S phase, and induce apoptosis.