We recently demonstrated that the N-acyl-homoserine lactone [autoinducer (AI)-1] and LuxS (AI-2)-based quorum-sensing (QS) systems exerted positive and negative regulation, respectively, on the virulence of a diarrhoeal isolate SSU of Aeromonas hydrophila. However, the role of a newly identified, two-component-based QseBC QS system in the regulation of bacterial virulence in general is not well understood, with only a limited number of studies showing its function in bacterial pathogenesis. In this report, we identified and characterized the QseBC QS system in A. hydrophila SSU and found that, as was the case with enterohaemorrhagic Escherichia coli, the open reading frames for the qseB (the response regulator) and qseC (the sensor histidine kinase) genes overlapped by 4 bp at the ATGA motif. Our data provide evidence that deletion of the qseB gene from A. hydrophila resulted in attenuation of bacterial virulence in a septicaemic mouse model of infection and diminished swimming and swarming motility, and the mutant bacteria formed denser biofilms compared with those from the parental strain of A. hydrophila. The decrease in the virulence of the A. hydrophila ΔqseB mutant correlated with reduced production of protease and the cytotoxic enterotoxin, which has associated haemolytic activity. The swimming and swarming motility, haemolytic activity, protease production and biofilm formation were restored in the qseBC-complemented strain to a level similar to that of the wild-type A. hydrophila SSU. Our study is the first, to our knowledge, to report a functional QseBC QS system in A. hydrophila which may be linked to AI-1 and AI-2 QS systems in modulating bacterial virulence, possibly through the cyclic diguanosine monophosphate.