Nitric oxide (NO) is produced as an innate immune response against microbial infections.SalmonellaTyphimurium (S. Typhimurium), the major causative pathogen of human gastroenteritis, induces more severe systemic disease in mice. However, host factors contributing to the difference in species-related virulence are unknown. Here, we report that host NO production promotesS. Typhimurium replication in mouse macrophages at the early infection stage by activatingSalmonellapathogenicity island-2 (SPI-2). The NO signaling-induced SPI-2 activation is mediated by Fnr and PhoP/Q two-component system. NO significantly inducedfnrtranscription, while Fnr directly activatedphoP/Qtranscription. Mouse infection assays revealed a NO-dependent increase in bacterial burden in systemic organs during the initial days of infection, indicating an early contribution of host NO to virulence. This study reveals a host signaling-mediated virulence activation pathway inS. Typhimurium that contributes significantly to its systemic infection in mice, providing further insights intoSalmonellapathogenesis and host–pathogen interaction.