Limosilactobacillus fermentum is known to exert immunomodulatory effects. In this study, we elucidated the immunoenhancing effects and mechanisms of a novel strain of Limosilactobacillus fermentum ATG-V5. Our findings demonstrate that ATG-V5 regulates the activation and proliferation of natural killer (NK) and T cells. It induced activation effects such as increased nitric oxide (NO) production, phagocytic capacity, and cytokine generation in Raw264.7 cells, which were confirmed to occur through the ERK and pp65 pathways. Furthermore, using a cyclophosphamide (CPP) mouse model, we demonstrated the immunomodulatory efficacy of ATG-V5, including enhanced activation of NK cells, increased levels of antibodies and cytokine secretion, in mice with immunosuppression induced by CPP. Additionally, by analyzing the gut bacterial community in the mouse model, we confirmed the ability of ATG-V5 consumption to regulate the gut microbiota, thereby substantiating the potential of this strain as a promising immunoenhancing agent.