The nitrite (NO2−) inhibition in anaerobic ammonium oxidation (anammox) process is widely reported. Here, the effects of three pyrolytic biochars (CS300, CS550 and CS800) were investigated to alleviate NO2− stress on anammox process under exposure of varied NO2−-N concentrations (70, 200, 400 and 600 mg L−1). No nitrite inhibition was observed at 70 mg N L−1. However, the total nitrogen removal efficiency (TNREs) decreased with NO2−-N concentration increased, while the biochar-amended groups achieved higher TNREs than the control (CK). At 200 mg N L−1, the TNREs were 60.2%, 99.0%, 98.5% and 86.6% for CK, CS300, CS550 and CS800, respectively. At 400 mg N L−1, the TNREs were 23.3%, 56.0%, 37.1% and 29.7% for CK, CS300, CS550 and CS800, respectively. At 600 mg N L−1 in which severe inhibition was observed, the TNREs were increased by 231% (p = 0.002), 149% (p = 0.014), and 51.0% (p = 0.166) for CS300, CS550 and CS800, respectively, as compared to CK, with the corresponding specific anammox activity increased by 3.1-, 2,0- and 1.1-folds, respectively. CS300 enriched the relative abundance of Candidatus Kuenenia and increased the gene copies of functional genes (hzsA, hdh, nirS and nirK). Besides, CS300 effectively alleviated the suppression of three membrane-associated enzyme complexes for anammox electron transport chain, indicating the possible contribution of redox-active moieties of CS300 to energy conversion metabolism for mitigating the NO2−-N inhibition. This study provided an effective strategy for alleviating NO2−-N stress by applying an environmentally compatible material (biochar) on anammox process.