This study aimed to investigate the importance of COD (chemical oxygen demand) and ratio of COD and nitrogen (COD/N) in influencing the dominance of anammox pathway to N-removal in anammox systems, which had been widely researched and results were not yet conclusive. Results showed that N-removal efficiency increased with increasing organic substrate, while the anammox contribution to N-removal decreased as confirmed by isotope labelling technique. Excessively high TN (total nitrogen) concentrations were detrimental to N-removal, and TN of 600 mg L^-1 was optimized. Specific COD of 300 mg L^-1 (a threshold value above which anammox was less active) was synergistic for N-removal. Moreover, Illumina sequencing and qPCR techniques uncovered that while the microbial community composition was relatively stable for all treatments, abundances of denitrifier were positively correlated with increase of COD, which was counter-productive for anammox abundance. Structure equation model indicated that COD was more important with respect to maintain the anammox stability than the COD/N ratio. Furthermore, experiment and model fittings revealed that anammox contributed more than 80% of N-removal when COD was below 55.7 mg L^-1, and approximately 50% at 220–300 mg L^-1 COD, respectively. These data formed a reference for regulation of anammox systems in real-world applications.