Background and Objective: With expansion of nanotechnology, nano-hydroxyapatite particles are used in medicine and dentistry fields. The biological effects of these particles are not completely defined. The aim of this study was to determine the cytotoxicity of nano-hydroxyapatite on gingiva-derived fibroblasts.Materials and Methods: Various concentrations of nano-hydroxyapatite from 2 to 0.002 mg/ml were treated on gingiva-derived fibroblast cell line (HGF2) in 24, 48 and 72 hours. MTT and LDH methods were used for evaluation of cytotoxic effect.Results: MTT method: doses higher than 5 and 2 mg/ml in 24 hours, doses higher than 0.2, 2 and 5 mg/ml and lower than 0.01, 0.02, 0.05, 0.002 and 0.005 in 48 hours and doses higher than 0.5, 1, 2, and 5 mg/ml and doses lower than 0, 0.01, 0.02, 0.002 and 0.005 mg/ml in 72 hours caused significant decreases in OD and absorbance of fibroblasts. LDH method: significant increase of LDH release noticed with 5 mg/ml dose in 24 hours, 1, 2, 5 mg/ml doses in 48 hours and 5 mg/ml dose in 72 hours. LDH release decreased with 0.02 mg/ml and 0.01 mg/ml in 24 and 48 hours, respectively. The IC50 was about 6 mg/ml in 24, 48 and 72 hours.Conclusion: In 24 hours and after that, high doses of nano-hydroxyapatite have cytotoxic effect on gingival-derived fibroblasts.