The aim was to characterize by atomic force microscopy (AFM) the nanostructure of human dentin surfaces affected by dental fluorosis (DF) before and after phosphoric acid etching. This study included 240 human dentin samples classified according to the severity of DF, which were divided into four groups using the Thylstrup-Fejerskov Index (TFI). Samples were analyzed by AFM before and after acid etching for 15, 30, and 60 s. The roughness (R(a)) for healthy dentin, and dentin with mild, moderate, and severe fluorosis were 440 nm, 442 nm, 445 nm, and 449 nm, respectively. After 15, 30, and 60 s of acid etching, all healthy and fluorotic dentin samples increased in roughness (p<0.05). The diameter of dentinal tubule orifices (D(t)) in healthy human dentin increased after acid etching for 60 s. We conclude that effective etching times are 15 s for healthy and mild dentin fluorosis, 30-s for moderately fluorosed dentin, and 45-60 s for severe fluorotic dentin.