Bacteria in biofilms are embedded in extracellular matrix and display low metabolic activity, partly due to insufficient diffusive exchange of metabolic substrate. The extracellular matrix and low metabolic activity both contribute to the high antibiotic tolerance-the hallmark of biofilm bacteria. The second messenger molecule, c-di-GMP, regulates biofilm development in Pseudomonas aeruginosa, where high internal levels lead to biofilm formation and low levels are associated with planktonic bacteria. Using a microcalorimetric approach, we show that c-di-GMP signaling is a major determinant of the metabolic activity of P. aeruginosa, both in planktonic culture and in two biofilm models. The high c-di-GMP content of biofilm bacteria forces them to rapidly spend a large amount of energy on the production of exopolysaccharides, resulting in a subsequent low metabolic state. This suggests that the low metabolic state of bacteria in mature biofilms, to some extent, is a consequence of a c-di-GMP-regulated survival strategy.