Abstract: Using the method of anaerobic glove chamber culture, active anaerobic bacteria were successfully enriched and cultivated in brown coal samples collected from Zhaotong Basin, Yunnan Province. Simulation experiments of biogenic gas for 60 days were further conducted to analyze the generation regularities, substance compositions and genetic mechanism of biogenic gas. The results showed that in all three brown coal samples there existed active anaerobic bacteria, among which cellulose decomposition bacteria were in majority and sulfate-reducing bacteria were very few, which means that the biochemical circumstances in the brown coal-seams of Zhaotong Basin were reductive and favourable for the growth of methanogen. The biogenic gas generation experienced through three stages: the very slow growing stage, the evidently increasing stage and the slowing down again stage, which indicated that methanogen had high density and activity in the second stage after the slow reproduction first stage. It is considered that the differences in gas generation amount and processes among three brown coal samples were probably resulted from the initial quantities of the active methanogen and maceral composition in the coal. It is proposed that there were at least two peaks of biogenic gas generation from the brown coal. Stimulated biogenic gas was mostly composed of CH4 and CO2, in which CH4 was predominant and heavy hydrocarbons were hardly detected. All the mean values of δ13C1 and δD in the methane produced from three coal samples were found to be in the normal distribution ranges compared to those of biogenic methane.