Actinobacillus pleuropneumoniaeis an important swine respiratory pathogen. Previous studies have suggested that growth as a biofilm is a natural state ofA. pleuropneumoniaeinfection. To understand the survival features involved in the biofilm state, the growth features, morphology and gene expression profiles of planktonic and biofilmA. pleuropneumoniaewere compared.A. pleuropneumoniaein biofilms showed reduced viability but maintained the presence of extracellular polymeric substances (EPS) after late log-phase. Under the microscope, bacteria in biofilms formed dense aggregated structures that were connected by abundant EPS, with reduced condensed chromatin. By construction of Δpgaand ΔdspBmutants, polymeric β-1,6-linked N-acetylglucosamine and dispersin B were confirmed to be critical for normal biofilm formation. RNA-seq analysis indicated that, compared to their planktonic counterparts,A. pleuropneumoniaein biofilms had an extensively altered transcriptome. Carbohydrate metabolism, energy metabolism and translation were significantly repressed, while fermentation and genes contributing to EPS synthesis and translocation were up-regulated. The regulators Fnr (HlyX) and Fis were found to be up-regulated and their binding motifs were identified in the majority of the differentially expressed genes, suggesting their coordinated global role in regulating biofilm metabolism. By comparing the transcriptome of wild-type biofilm and Δpga, the utilization of oligosaccharides, iron and sulfur and fermentation were found to be important in adhesion and aggregation during biofilm formation. Additionally, when used as inocula, biofilm bacteria showed reduced virulence in mouse, compared with planktonic grown cells. Thus, these results have identified new facets ofA. pleuropneumoniaebiofilm maintenance and regulation.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13567-023-01173-x.