Zymomonas mobilis is an emerging chassis for being engineered to produce bulk products due to its glycolysis through the Entner-Doudoroff pathway with less ATP produced for lower biomass accumulation and higher yields with targeted products. When self-flocculated, the bacterial cells are more productive and tolerant to stresses for high product titers, but this morphology needs to be controlled properly to avoid internal mass transfer limitation associated with strong flocculation. Herewith we explored the regulation of cyclic diguanosine monophosphate (c-di-GMP) on self-flocculation of the bacterial cells through cellulose biosynthesis. While ZMO1365 and ZMO0919 with GGDEF domains for diguanylate cyclase activities catalyze c-di-GMP biosynthesis, ZMO1487 with an EAL domain for phosphodiesterase activities catalyzes c-di-GMP degradation, but ZMO1055 and ZMO0401 contain the dual domains with phosphodiesterase activities predominated. Since c-di-GMP is synthesized from GTP, the intracellular accumulation of this signal molecule through deactivating the activity of phosphodiesterase is preferred for activating cellulose biosynthesis to flocculate the bacterial cells, since such a strategy exerts less perturbance on intracellular processes regulated by GTP. These discoveries are significant not only for engineering unicellular Z. mobilis strains with the self-flocculating morphology to boost production, but also for understanding mechanism underlying c-di-GMP biosynthesis and degradation in the bacterium.