Nucleotide signalling pathways in bacteria are key regulators of cellular responses under external stimuli. Cyclic di-adenosine monophosphate (c-di-AMP) is one of recently discovered messengers in a broad range of bacteria that regulates numerous important cellular processes, however, signals that trigger its accumulation or depletion are poorly understood. Levels of c-di-AMP have been found to inversely correlate with bacterial osmoresistance due to inhibition of potassium and compatible solute uptake systems. In this study, a genetic screen revealed several gain-of-function suppressor mutations in a Kup family potassium transporter that restored osmoresistance in a high c-di-AMP phosphodiesterase Lactococcus lactis mutant. Surprisingly these mutations led to significantly elevated c-di-AMP levels. Constitutive potassium uptake was found to trigger c-di-AMP accumulation, possibly in response to increased turgor pressure. In agreement with this, c-di-AMP levels in resting and energised Lactococcus, Lactobacillus, Staphylococcus and Listeria were found to be rapidly responsive to external osmolarity changes. These results demonstrate a feedback loop exists whereby c-di-AMP regulates osmoresistance and is in turn modulated by cellular osmotic perturbations in bacteria.