The valuable minerals in copper-nickel sulfide ore can easily be oxidized, leading to the reduction of their flotation recovery and a difficulty in separating them from gangue. In order to solve the problem, the reaction mechanism of the octanohydroxamic acid (OHA) on oxidized pyrrhotite was revealed through micro-flotation, adsorption tests, zeta potential measurements, and X-ray photoelectron spectroscopy (XPS) analysis. The results show that this is a feasible way to find a suitable collector that can directly react to oxidation products on the surface of pyrrhotite. OHA can efficiently reclaim oxidized pyrrhotite and achieve the selective separation of a pyrrhotite-serpentine mixture in a weak alkaline environment. The adsorption tests, zeta potential measurements, and XPS analyses show that OHA can interact with an oxidized pyrrhotite surface, and the interaction between OHA and serpentine is very weak. The XPS analyses indicate that the OHA collector can chelate with Fe(OH)3 on the surface of oxidized pyrrhotite and form an “O, O” five-ring chelate. At the same time, the OHA collector may compete with the hydroxyl groups of hydrophilic substances on the mineral surface to produce hydrophobic products and reduce the hydrophilic substances on the mineral surface.