The coal after low-temperature pyrolysis is more prone to self-heating and spontaneous combustion. To study the original heat source, this paper conducted sequentially isothermal pyrolysis, room-temperature oxidation and low-temperature re-oxidation on four different coal samples. Formation laws of active sites were studied through gas emission rules during isothermal pyrolysis of coal. Besides, the influence of pyrolyzed coal at low temperatures on spontaneous combustion in following mining process was stimulated by the subsequent room-temperature oxidation and low-temperature re-oxidation of the coal. Experimental results reveal that during low-temperature pyrolysis of coal samples, oxygen-containing functional groups are decomposed. They produce numerous active sites which are stabilized in anaerobic conditions. These sites are so active that they can be oxidized at room temperature to release massive CO and CO2 and huge heat. Therefore, the viewpoint of room-temperature oxidation of active sites is proposed. The exothermic oxidation of active sites at room temperature is the initial heat source of self-heating and spontaneous combustion of the pyrolyzed coal. Furthermore, room-temperature oxidation of the pyrolyzed coal presents object proof of direct burn-off reaction, which is of great significance to the interpretation of coal spontaneous combustion mechanism.