Heat shock proteins (HSPs) play important biological roles, and they are implicated in bacterial response to environmental stresses and in pathogenesis of infection. The role of HSPs in P. aeruginosa, however, remains to be fully elucidated. Here, we report the unique role of HSP DnaJ in biofilm formation and pathogenicity in P. aeruginosa. A dnaJ mutant produced hardly any pyocyanin and formed significantly less biofilms, which contributed to decreased pathogenicity as demonstrated by reduced mortality rate in a Drosophila melanogaster infection model. The reduced pyocyanin production in the dnaJ mutant was a result of the decreased transcription of phenazine synthesis operons including phzA1, phzA2, phzS, and phzM. The reduction of biofilm formation and initial adhesion in the dnaJ mutant could be reversed by exogenously added pyocyanin or extracellular DNA (eDNA). Consistent with such observations, absence of dnaJ significantly reduced the release of eDNA in P. aeruginosa and addition of exogenous pyocyanin could restore eDNA release. These results indicate dnaJ mutation caused reduced pyocyanin production, which in turn caused the decreased eDNA, resulting in decreased biofilm formation. DnaJ is required for pyocyanin production and full virulence in P. aeruginosa; it affects biofilm formation and initial adhesion via pyocyanin, inducing eDNA release.