The arginine deiminase (ADI) pathway catalyses the degradation of L-arginine via the intermediate L-citrulline into L-ornithine, ammonia, and CO2. This constitutes an important metabolic feature of some lactic acid bacteria (LAB) in their response to acid stress. We investigated the capacity of four thermophilic starters to degrade arginine via the ADI pathway and studied their influence on the ripening and quality of experimental Gruyere cheeses. One of the starters could not degrade arginine, while another had an impaired ADI pathway and formed mainly citrulline. In cheeses produced with the two starters exhibiting a fully functional ADI pathway, ornithine content, pH-value, and CO2 and eye formation were significantly increased, indicating that the quality and ripening of the cheeses were affected by ADI metabolism. This is the first study investigating thermophilic starter cultures that contain Lactobacillus delbrueckii subsp. lactis with a functional ADI pathway converting arginine into ornithine and CO2 in cheese.