This study investigated the influence of nitrate on aerobic granular sludge (AGS) granulation. The introduction of nitrate at 5, 15 and 20 mg L−1 promoted AGS granulation, and the promoting effect was positively correlated with nitrate concentrations. Meanwhile, exogenous nitrate significantly increased denitrification rate in the AGS system. However, granular disintegration appeared at a long-term addition of nitrate. An in-deep analysis showed that nitrate stimulated the secretion of extracellular polymeric substances (EPS), especially the content of proteins, which might be the main reason for the AGS granulation. However, the rapid and excessive increase in EPS might cause granular disintegration, as excessive EPS blocked the transmission of substrates, leading to the increase of dead cells in the granules. Besides, nitrate also altered the hydrophobicity of EPS and the content of α-helix, 3-turned helix and polymeric chain that favored aggregation, which also affected AGS granulation. From the microbial community level, nitrate induced the enrichment of denitrifying bacteria, including those that also functioned as EPS producers, such as Micropruina and Flavobacterium, resulting in the rapid increase of functional enzymes associated with amino acid synthesis, thereby promoting the secretion of proteins in EPS. Conversely, disintegration caused by mass transfer blockage might lead to the loss of EPS producing bacteria and subsequent decrease in EPS content, further accelerating granular disintegration. Display omitted •AGS granulation was improved with the increase of nitrate from 5 to 20 mg L−1.•Granule disintegration appeared under a long-term effect of 5–20 mg L−1 nitrate.•The increase in nitrate promoted EPS, but excessive EPS might lead to disintegration.•Nitrate altered the hydrophobicity and the content of proteins secondary structures.•Nitrate stimulated denitrifiers with EPS producing ability.