Microgravity is a major environmental factor of space flight that triggers dysregulation of the immune system and increases clinical risks for deep-space-exploration crews. However, systematic studies and molecular mechanisms of the adverse effects of microgravity on the immune system in animal models are limited. Here, we establish a ground-based zebrafish disease model of microgravity for the research of space immunology. RNA sequencing analysis demonstrates that the retinoic-acid-inducible gene (RIG)-I-like receptor (RLR) and the Toll-like receptor (TLR) signaling pathways are significantly compromised by simulated microgravity (Sμg). TRIM25, an essential E3 for RLR signaling, is inhibited under Sμg, hampering the K63-linked ubiquitination of RIG-I and the following function-induction positive feedback loop of antiviral immune response. These mechanisms provide insights into better understanding of the effects and principles of microgravity on host antiviral immunity and present broad potential implications for developing strategies that can prevent and control viral diseases during space flight. Display omitted •Simulated microgravity attenuates the antiviral innate immune responses•Simulated microgravity hampers TRIM25-mediated K63-linked ubiquitination of RIG-I•TRIM25 function-induction positive feedback loop is essential in antiviral immunity•Reduced TRIM25 expression under simulated microgravity interferes with the feedback loop Using a ground-based zebrafish model, Zhu et al. show that simulated microgravity suppresses TRIM25-mediated K63-linked ubiquitination of RIG-I and the subsequent function-induced positive feedback loop of the immune response. These findings reveal the effects of microgravity on immunity and highlight potential directions for disease control during space flight.