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The gram‐negative plant pathogen Xanthomonas oryzae pv. oryzae (Xoo) is able to infect the host rice and effectively colonize in vascular tissues. The type IV pilus (T4P) is one of the major virulence factors playing an important role in migration of Xoo through host vascular tissues. Here, we identified PilN, a T4P alignment subcomplex protein, which is involved in regulation of swimming motility, and analysed its contribution to bacterial surface‐associated behaviours and virulence. We found that the pilN deletion mutant exhibited dramatically reduced twitching motility and scarcely detectable levels of T4P major pili PilA, as well as enhanced biofilm formation and exopolysaccharide (EPS) production. In addition, deletion of the pilN gene in Xoo resulted in impaired virulence in host rice and attenuated type III secretion system (T3SS) genes expression, which is independent of PilA assembly. Expression of the relevant pilN gene in trans was capable of restoring twitching motility and biofilm formation to the wild‐type levels in the pilN mutant but partially recovering EPS production and virulence. Moreover, the expression of trh and xrvA genes, which encode the HrpG positive regulators, was decreased in the pilN mutant. Our results suggest that PilN executes versatile functions in bacterial virulence and cell surface‐associated behaviours.