The depression of hematite in the reverse flotation system of oleate-starch-hematite was investigated at the atomic level by atomic force microscopy (AFM) combined with density functional theory (DFT). AFM imaging and cross-sectional imaging show that the adsorption configurations of oleate and starch on hematite were in shuttle and flake patterns, respectively, resulting in a greater adsorption thickness and adsorption area for starch than for oleate on the hematite surface. After functioned by starch, hematite was almost fully covered by starch so that there was no sufficient space for the adsorption of oleate. Corn starch was also detected aggregating fine hematite particles just like a flocculant. In the DFT study of the system, convergence tests were conducted to get the optimized hematite crystal geometry. The calculated interaction energies and Mulliken populations verified the favorable adsorption of oleate and starch on the hematite surface. Results indicated that covalent bonds formed between oleate and hematite, as well as between starch and hematite. However, in the oleate-starch-hematite system, the calculations showed that the distance between oleate and hematite in the presence of starch was too long to allow the adsorption of oleate. As for the water effect on the depression of hematite, the water molecules acted as a bridge between starch and the hematite surface by hydrogen bonds and were accompanied by chemical bonds between hematite surfaces and starch, which led to the depression of hematite. Therefore, in reverse flotation, oleate is hindered from adsorbing on the starch-covered hematite surface. The work presented here has profound implications for future studies of the depression of hematite, and may help solve the problem of screening and design of depressant of hematite.