Introduction: Interproximal enamel reduction is a part of the orthodontic treatment as a method of space generation in addition to other vast indications. Some studies found that different techniques might impose changes to the enamel surface that alter its topography, which in turn might influence its integrity and susceptibility to caries. Polishing, however, after this procedure is thought to be helpful to reduce these adverse effects. Aim: To evaluate the nano-topography of the enamel surfaces after interproximal reduction (IPR) and determine its influence on enamel surface roughness and examine the need for polishing to minimise these influences, when combined with topical fluoride application. Methods: A total of 60 proximal surfaces of 40 extracted maxillary premolars (10 premolars left unprepared as the control group) were reduced with different stripping instruments (discs, burs and manual strip system). The surface roughness of enamel was analysed with an atomic force microscope to determine the results quantitatively as well as qualitatively on the nanoscopic scale. One of each proximal surface was followed by polishing and fluoride varnish after the reduction. Results: The results showed that surface roughness was increased in all groups without polishing. The greatest mean roughness was recorded for the disc group (212 ±125.7), followed by the bur group (172 ±93.1) and manual strips (153.8±106.7). The difference between the groups, however, was not significant for both mean roughness ( P = 0.656) and height ( P = 0.737). The parameters were decreased after polishing in all groups but the difference between methods was not significant for both parameters ( P = 0.946 and P = 0.849); however, the mean height was reduced to nearly half the reading in the bur and manual strip method. The disc group only showed a statistically significant decrease in surface roughness with polishing ( P < 0.05). All other results were not significant. Conclusion: All methods of interproximal reduction do not influence enamel surface nanotopography significantly with and without polishing. Polishing resulted in significant reduction of surface roughness only in the disc group.