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Listeriosis is a zoonotic disease caused by Listeria monocytogenes (Lm), posing a significant threat to the breeding industry and public health. Ruminant livestock are particularly susceptible to Lm,  thus effective strategies are needed for controlling ovine listeriosis. In this study, we developed two inactivated vaccines and evaluated their efficacy against Lm infection in murine and ovine models. We inactivated the Lm serotype 4h XYSN strain and adjuvanted it with water-in-oil ISA 61 VG (61 VG-AIV) or aluminum (Al-AIV). Pathological observations confirmed the safety of both vaccines in mice and sheep. The immunological assays demonstrated that, compared with the Al-AIV, the 61 VG-AIV induced higher levels of Lm-specific antibodies and proinflammatory cytokines, suggesting that the ISA 61 VG adjuvant has superior immunostimulatory effects compared with the alum adjuvant. 61 VG-AIV elicited greater immunoprotection than Al-AIV (83.4% vs. 50%) against serotype 4h Lm strain challenge in mice. Additionally, 61 VG-AIV afforded cross-protection against challenges with serotypes 1/2a, 1/2b, and 4b Lm strains. Importantly, high immunoprotection in sheep was conferred by the 61 VG-AIV group (83.4%). Taken together, our findings demonstrate that the ISA 61 VG adjuvant contributes to enhancing the humoral and cellular immune responses of inactivated Lm, and 61 VG-AIV is a promising vaccine candidate for the prevention and control of animal listeriosis. This research lays a solid foundation for its application in veterinary medicine.