Pseudomonas aeruginosa and other Gram-negative bacteria release membrane vesicles (MVs) from their surfaces, and MVs have an ability to interact with bacterial cells. Although it has been known that many bacteria have mechanisms that control their phenotypes with the transition from exponential phase to stationary phase, changes of properties in released MVs have been poorly understood. Here, we demonstrate that MVs released by P. aeruginosa during the exponential and stationary phases possess different physiochemical properties. MVs purified from the stationary phase had higher buoyant densities than did those purified from the exponential phase. Surface charge, characterized by zeta potential, of MVs tended to be more negative as the growth shifted to the stationary phase, although the charges of PAO1 cells were not altered. Pseudomonas quinolone signal (PQS), one of the regulators related to MV production in P. aeruginosa, was lower in MVs purified from the exponential phase than in those from the stationary phase. MVs from the stationary phase more strongly associated with P. aeruginosa cells than did those from the exponential phase. Our findings suggest that properties of MVs are altered to readily interact with bacterial cells along with the growth transition in P. aeruginosa.