Despite its significant potential in various disease treatments and diagnostics, microbiotherapy is consistently plagued by multiple limitations ranging from manufacturing challenges to in vivo functionality. Inspired by the strategy involving nonproliferating yet metabolically active microorganisms, we report an intracellular gelation approach that can generate a synthetic polymer network within bacterial cells to solve these challenges. Specifically, poly(ethylene glycol dimethacrylate) (PEGDA, 700 Da) monomers are introduced into the bacterial cytosol through a single cycle of freeze-thawing followed by the initiation of intracellular free radical polymerization by UV light to create a macromolecular PEGDA gel within the bacterial cytosol. The molecular crowding resulting from intracytoplasmic gelation prohibits bacterial division and confers robust resistance to simulated gastrointestinal fluids and bile acids while retaining the ability to secrete functional proteins. Biocompatibility assessments demonstrate that the nondividing gelatinized bacteria are effective in alleviating systemic inflammation triggered by intravenous Escherichia coli injection. Furthermore, the therapeutic efficacy of gelatinized Lactobacillus rhamnosus in colitis mice provides additional support for this approach. Collectively, intracellular gelation indicates a universal strategy to manufacture next-generation live biotherapeutics for advanced microbiotherapy.