Staphylococcus aureus is able to cause chronic and acute infections. Most strains form biofilms. SA0701 (gdpS) codes for a protein with a GGDEF domain that catalyses cyclic-di-GMP production in many bacteria. In S. aureus, the role of GdpS remains poorly understood. We deleted gdpS in three independent S. aureus isolates. The modifications on the transcriptomic and on the proteomic levels were assessed. This study suggests that the role of GdpS may be strain-dependent. Dynamic imaging during biofilm development showed that GdpS inhibited biofilm formation in some of the backgrounds tested, but had no effect on bacterial survival. We found an increased susceptibility to low temperatures in the mutant strains. GdpS is not responsible for c-di-GMP production but acts as a partner for genes involved in adaptative metabolism. GdpS might also contribute to virulence and may act as a sensor protein. Finally, GdpS decreased the amount of eDNA affecting biofilm stability.