Abstract: To explore the optimal combination of lyoprotective agents for effectively enhancing the viability of Akkermansia muciniphila (Akk) and elucidating the underlying mechanisms.This study aimed to optimize lyoprotectant formulations using single-factor experiments and response surface methodology for freeze-drying (lyophilization) of Akk DSM 22959. Bacterial powder was analyzed using Fourier-transform infrared spectroscopy (FTIR), glass transition temperature (Tg), and scanning electron microscopy (SEM) and by assessing Na⁺/K⁺ ATPase activity and membrane integrity. The formulation sustained Na⁺/K⁺ ATPase activit and achieved a post-lyophilization viability of 83.41%±0.41%. The survival rate of Akk powder was still higher after storage at −20 ℃ and 4 ℃ for 60 days. The optimal lyoprotectant (composed of skim milk powder, maltodextrin, sodium glutamate, and tremella polysaccharides) formed a protective matrix that encapsulated the Akk cells. This matrix preserved membrane integrity, maintained physiological morphology, and elevated Tg, thereby substantially reducing cryo-injury during lyophilization.These findings provide critical mechanistic insights into lyoprotection strategies and establish a scalable protocol for industrial production of live Akk biotherapeutic products.