High sulfur content in excess sludge impacts the production of biomethane during anaerobic digestion, meanwhile leads to hydrogen sulfide (H2S) formation in biogas. Effect of initial sludge pH on H2S formation during batch mesophilic anaerobic digestion of slaughterhouse wastewater sludge was studied in this paper. The results demonstrated that when the initial sludge pH increased from 6.5 to 8.0, the biogas production increased by 10.1%, the methane production increased by 64.1%, while the H2S content in biogas decreased by 44.7%. The higher initial sludge pH inhibited the competition of sulfate-reducing bacteria with methane-producing bacteria, and thus benefitted the growth of methanogens. Positive correlation was found between the relative abundance of Desulfomicrobium and H2S production, as well as the relative abundance of Methanosarcina and methane production. More sulfates and organic sulfur were transferred to solid and liquid rather than H2S formation at a high initial pH.