BACKGROUND Interproximal reduction (IPR) removes enamel and leaves grooves and furrows on the tooth surface, which may increase the risk of caries. The aims of this study were to assess the nanotopography of enamel surfaces produced by the most commonly used IPR instruments and to evaluate the effect of polishing after IPR. METHODS Enamel slabs were cut from the interproximal surfaces of healthy premolars and then treated with diamond burs, strips, or discs, or Sof-Lex polishing discs (3M ESPE, St Paul, Minn). All samples were cleaned by sonication in distilled water. The control group had no IPR performed and was subjected only to cleaning by sonication. The enamel surfaces were assessed using atomic force microscopy. RESULTS The IPR instruments all produced surfaces rougher than the control sample; however, the samples that received polishing with Sof-Lex discs after enamel reduction were smoother than untreated enamel (P <0.05 for all comparisons). The larger grit medium diamond burs and medium strips generated rougher enamel surfaces than their smaller grit counterparts: fine diamond burs and fine strips (P <0.001). The difference in roughness generated by mesh and curved disks was not statistically significant (P = 0.122), nor was the difference caused by fine strips and mesh discs (P = 0.811) or by fine strips and curved discs (P = 0.076) (surface roughness values for medium bur, 702 ± 134 nm; medium strip, 501 ± 115 nm; mesh disc, 307 ± 107 nm; fine bur, 407 ± 95 nm; fine strip, 318 ± 50 nm; curved disc, 224 ± 65 nm). The smoothest surfaces were created by use of the entire series of Sof-Lex polishing discs after the enamel reduction (surface roughness, 37 ± 14 nm), and these surfaces were significantly smoother than the control surfaces (surface roughness, 149 ± 39 nm; P = 0.017). CONCLUSIONS Different IPR instruments produced enamel surfaces with varied nanotopography and different degrees of roughness. Enamel surfaces treated with diamond-coated burs were the roughest, followed by diamond-coated strips and diamond coated discs. Polishing with Sof-Lex polishing discs after IPR reduced the enamel surface roughness, and this surface was even smoother than untreated enamel.