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Abstract Pili have been demonstrated to contribute to the pathogenicity of many bacterial pathogens. Flp pilus encoded by the tad locus belongs to the type IVb pilus. Our previous study has revealed that the intact tad locus is essential for Flp pilus formation in Actinobacillus pleuropneumoniae , a very important porcine respiratory pathogen. To further investigate the functions of Flp pilus in A. pleuropneumoniae pathogenesis, the flp1 and tadD single deletion mutants were constructed by homologous recombination. Both of the mutant strains lost pilus on their cell surfaces. The abilities of biofilm formation, cell adhesion, resistance to phagocytosis, survival in swine whole blood, and in vivo colonization of the two mutants were significantly reduced compared with those of the parental strain. The corresponding complemented strains recovered the phenotypes. These results demonstrated that flp1 and tadD were essential for the biosynthesis of Flp pilus and that the pilus played important roles during infection of A. pleuropneumoniae. Highlights • The genes flp1 and tadD are essential for biosynthesis of A. pleuropneumoniae Flp pilus. • A. pleuropneumoniae Flp pilus is important for biofilm formation. • A. pleuropneumoniae Flp pilus is involved in adherence and anti-phagocytosis in vitro. • Flp pilus plays essential roles in A. pleuropneumoniae colonization in vivo. [ABSTRACT FROM AUTHOR]