Effects of nitrate addition on pH and biogas production in the anaerobic straw and manure fermentation system co-cultured with Alcaligenes faecalis (Af) and Bacillus thuringiensis (Bt) were investigated. The acidification effect was occurred in the anaerobic straw and manure fermentation system, and the pH value varied from 5.2 to 5.5. Although the fermentation system co-cultured with Af and Bt enhanced the pH by 2.9%-13.6%, the pH value was still below 6. After 168 h anaerobic fermentation, the biogas production, pH value and volatile fatty acid (VFA) content of the fermentation system co-cultured Af/Bt with the inoculum  ratio of 1/2 and nitrate addition (2g/L) was increased by 62.8%, 22.8% and decreased by 13.0%, responding to 350mL, 7.12g/L and 2.54g/L, respectively, compared with the control (the fermentation system co-cultured Af/Bt with the inoculation volum ratio of 1/2). The results suggested that nitrate addition influenced the anaerobic straw and manure fermentation system remarkably, and the ammonifying bacteria such as Af and Bt did not show the ability of nitrate respiration with the unavailable nitrate. Therefore, the inoculation with ammonifying bacteria alone did not enhance the pH of the system. The nitrate addition in the system lessened the VFA production, promoted the pH value of the anaerobic straw and manure fermentation system and improved the biogas synthesis efficiency in the end. Meanwhile, the results also suggested the bottleneck of the anaerobic straw and manure fermentation system for biogas was duo to the low pH value as opposed to the VFA synthesis efficiency.