Mineral surface wettability and its regulation by the adsorption of collectors have an important influence on the flotation performance. The adsorption behavior of C18 fatty acid with different unsaturation and its effect on rhodochrosite wettability was investigated with surface tension, contact angle, and atomic force microscopy (AFM) measurements. The results indicated that rhodochrosite hydrophobicity increased with the increasing concentration of fatty acid, along with the maximum contact angle (θmax) between hemimicelle concentration (HMC) and critical micelle concentration (CMC). Oleic acid (OA), linoleic acid (LA), and α-linolenic acid (ALA) had a higher θmax than stearic acid (SA), but the value decreased with the increase of C=C bond number. Besides, preferential adsorption of unsaturated fatty acids on the liquid-air interface can be attributed to the molecule’s steric hindrance resulting from C=C double bond, and the θ kept almost invariant with a higher value of ΓLG than ΓSL until HMC. The oriented monolayer and bilayer structure of fatty acids formed gradually on rhodochrosite surface with increasing concentration. However, the θmax may not necessarily correspond to the beginning of bilayer formation. Cylindrical monolayer and bilayer micelles of SA molecules were observed on rhodochrosite surface at HMC and CMC, respectively. While bilayer structures of unsaturated fatty acids formed before complete coverage of monolayer on rhodochrosite surface because of surface heterogeneity. This work provided a good understanding on the adsorption mechanism of fatty acid on rhodochrosite for flotation.