Pseudomonas aeruginosa is an opportunistic human pathogen that produces and secretes exopolysaccharides (EPS), in which cells are embedded to form a highly organized community structure called biofilm. Here, we characterized the role of cyclic diguanylate (c-di-GMP) and EPS (PEL) overproduction in the wspF mutant phenotypes of P. aeruginosa PA14 (wrinkly appearance, hyperadherence, impaired motilities, and reduced virulence in acute infections). We confirmed that the elevated c-di-GMP level plays a key role in all the wspF mutant phenotypes listed above, as assessed by ectopic expression of a c-di-GMP-degrading phophodiesterase (PvrR) in the wspF mutant. In contrast, PEL EPS, which is overproduced in the wspF mutant, was necessary for wrinkly appearance and hyperadherence, but not for the impaired flagellar motilities and the attenuated virulence of the wspF mutant. These results suggest that cdi- GMP affects flagellar motility and virulence, independently of EPS production and surface adherence of this bacterium.