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Sulfation roasting, a common activation technique, is a potential method for cleaner production of nickel from complex low-grade ores. In this study, nickel oxide–sulfide mixed ore concentrate was roasted with the addition of ammonium sulfate under a static air atmosphere, and the roasted products were leached by water, in order to evaluate the extraction of metals. The ammonium sulfate activation roasting was investigated thoroughly and systematically by thermogravimetry–differential scanning calorimetry, X-ray diffraction, and scanning electron microscopy. Particularly, the interface sulfation behavior and path were studied by the density functional theory (DFT) method. The results showed that a large amount of nonferrous metal sulfate (70% Ni, 89% Co, and 90% Cu) was generated, while iron was almost entirely transformed into iron oxide under appropriate roasting conditions of adding ammonium sulfate at a mass ratio of 200%, heating to 650 °C at 10 °C/min, and holding for 120 min. It was found that activation of ammonium sulfate can take two different paths: one in which ammonium sulfate directly reacts with raw ores below 500 °C and the other in which the SO2 decomposed from sulfates (ammonium sulfate, intermediate ammonium ferric sulfate, and ferric sulfate) reacts with the intermediate metal sulfides (NiS and Cu2S). The interface sulfation mechanism of NiS and Cu2S was investigated deeply by DFT method, which showed that there are two paths of sulfation for NiS or Cu2S, and both of them are thermodynamically favored. Thus, a thorough and systematic investigation of ammonium sulfate activation roasting of nickel oxide–sulfide mixed ore is provided; this might be a potential basis for future industrial applications of ammonium sulfate activation roasting techniques in complex mineral metallurgy.