Roughness of mineral particles affects bubble-particle interactions and particle flotation separation. In this study, magnesite was ground in stainless-steel disc and ceramic ball mills to produce flotation feed material of different surface nano-roughness, which was quantified using atomic force microscopy (AFM). The effect of surface roughness on the floatability of magnesite particles was carried out by a micro-flotation kinetic test using an XFG flotation machine. The flotation results demonstrated both higher flotation recovery and larger flotation rate constant for particles having larger nano-asperities of rough particle surface. Additionally, the difference in cumulative flotation recovery between magnesite particles with different nano-roughness characteristics systematically decreased with increasing collector (sodium oleate) concentration from 25 to 150 mg/L. Contrarily, a difference in the value of flotation kinetics constant for particles with larger and smaller asperities remained at a level of 0.2–0.3 min−1 for the entire range of collector concentration. The interaction energy between bubbles and rough magnesite particles with different nano-roughness was estimated using an extended DLVO (Derjaguin–Landau–Verwey–Overbeek) theory. The theoretical interaction energy points to lowering energy barrier when the magnesite particles are covered with 12 nm asperities as compared to 2 nm asperities. It is therefore hypothesized that the energy barrier is a primary cause for differences in flotation performance of particles decorated with nano-asperities of different dimensions.