Effect of bicarbonate concentration ([HCO₃^-]) on hydrogenotrophic methanogens (HM) and aceticlastic methanogens (AM) in anaerobic activated sludge was investigated with H₂/CO₂ and acetate as the sole carbon source, respectively. The mechanisms of bicarbonate's effect on methane formation was analysed based on the methane production efficiency, community structure of methanogens, reaction kinetics and thermodynamics. The results showed that [HCO₃^-] ranged from 0.05 to 0.20 mol/L could stimulate the activity of HM, and the higher hydrogen partial pressure, the lower [HCO₃^-] for the stimulation. An increased [HCO₃^-], on the contrary, would inhibit the methanogenesis of AM. Methanobacterium formicium and Methanosarcina mazei were identified as the dominated HM and AM, respectively, in the incubated anaerobic active sludge. It was found that the abundance of M. formicicum was enhanced by the increased [HCO₃^-] in the fermentation systems. The abundance of M. mazei was also improved by a [HCO₃^-] less than 0.10mol/L, but decreased with the [HCO₃^-] over than 0.15mol/L. Variation of [HCO₃^-] could significantly change the pH and Gibbs free energy of the methanogenic reactions in the fermentation processes, and the methanogenesis activities of HM and AM were further affected as a result.