The influence of conditioning on the flotation of pyrrhotite in the presence of chlorite was investigated through flotation tests, sedimentation tests, and X-ray photoelectron spectroscopy (XPS) analysis. The flotation results show that chlorite slimes dramatically impair the flotation of pyrrhotite. Sedimentation and flotation tests reveal that conditioning can effectively remove chlorite slimes from pyrrhotite surfaces, resulting in an enhanced flotation recovery of pyrrhotite. When mixed minerals were conditioned under the natural atmosphere, a faster conditioning speed and longer conditioning time decreased the flotation recovery of pyrrhotite. However, when mixed minerals were conditioned under a nitrogen atmosphere, a more intensive conditioning process provided better flotation results. XPS analyses illustrate that a faster conditioning speed and longer conditioning time under the natural atmosphere accelerates the oxidation of pyrrhotite, leading to a decrease in the flotation recovery of pyrrhotite.