Background: Influenza is a highly contagious respiratory disease that poses significant health and economic burdens. Mice are commonly used as animal models for studying influenza virus pathogenesis and the development of vaccines and drugs. However, the viral volume used for nasal inoculation varies substantially in reported mouse influenza infection models, and the appropriate viral dose is crucial for reproducing experimental results. Methods: Mice were inoculated with mouse lung-adapted strains of influenza virus A/Puerto Rico/8/34 (H1N1) via intranasal administration of 10 μL, 20 μL, and 40 μL at doses of 200 plaque-forming units (PFU) and 2000 PFU. This study investigated the impact of varying viral inoculum volumes on murine outcomes at identical doses and assessed the disparities across diverse dosage levels. Results: Regarding weight change trajectories, mortalities, lung tissue viral titers, and pathological manifestations, the group that received the 40 μL inoculation volume within the low-dose infection mice (200 PFU) manifested a statistically significant divergence from those inoculated with both the 10 μL and 20 μL volumes. Within the context of high-dose infections (2000 PFU), groups that received inoculation volumes of 20 μL and 40 μL exhibited marked disparities when compared to those receiving the 10 μL volume. Conclusions: Disparities in inoculation volume, even under uniform infection dosages, engender differential outcomes in pathogenicity. Of particular note, the viral replication efficacy at a 20 μL inoculation volume demonstrates conspicuous fluctuations across diverse infection dose regimens.