The physiology of animal cells is characterized by constantly changing environmental conditions and adapting cellular responses. Applied dynamic metabolic flux analysis captures metabolic dynamics and can be applied to industrially relevant cultivation conditions. We investigated the impact of glutamine availability or limitation on the physiology of CHO K1 cells in eight different batch and fed-batch cultivations. Varying glutamine availability resulted in global metabolic changes. We observed dose-dependent effects of glutamine in batch cultivation. Identifying metabolic links from the glutamine metabolism to specific metabolic pathways, we show that glutamine feeding results in its coupling to tricarboxylic acid cycle fluxes and in its decoupling from metabolic waste production. We provide a mechanistic explanation of the cellular responses upon mild or severe glutamine limitation and ammonia stress. The growth rate of CHO K1 decreased with increasing ammonia levels in the supernatant. On the other hand, growth, especially culture longevity, was stimulated at mild glutamine-limiting conditions. Flux rearrangements in the pyruvate and amino acid metabolism compensate glutamine limitation by consumption of alternative carbon sources and facilitating glutamine synthesis and mitigate ammonia stress as result of glutamine abundance.