Aeromonas hydrophila is the causative agent of motile Aeromonad septicemia in fish. Previous studies have shown that the myo-inositol metabolism is essential for the virulence of this bacterium. IolR is a transcription inhibitor that negatively regulates myo-inositol metabolic activity. While in the process of studying the inositol catabolism in A. hydrophila Chinese epidemic strain NJ-35, we incidentally found that ΔiolR mutant exhibited obvious autoaggregation and increased biofilm formation compared to the wild type. The role of surface proteins in A. hydrophila autoaggregation was confirmed by different degradation treatments. Furthermore, calcium promotes the formation of aggregates, which disappear in the presence of the calcium chelator EGTA. Transcriptome analysis, followed by targeted gene deletion, demonstrated that biofilm formation and autoaggregation caused by the inactivation of iolR was due to the increased transcription of a RTX-family adhesion gene, rmpA. Further, IolR was determined to directly regulate the transcription of rmpA. These results indicated that iolR is negatively involved in autoaggregation and biofilm formation in A. hydrophila, and this involvement was associated with its inhibition on the expression of rmpA.