The tumor necrosis factor-alpha (TNF-alpha) influences the ability to limit parasite infection but its over-production might result in inflammatory disorders. The level of Tnf-alpha gene expression could thus mediate a balance of tolerance/resistance to infections. This study focused on Puumala hantavirus (PUUV) infection in its rodent host, the bank vole (Myodes glareolus). In humans, PUUV is responsible of a mild form of hemorrhagic fever with renal syndrome, nephropathia epidemica (NE). The severity of NE is associated with an over-production of TNF-alpha. By contrast, PUUV infection in bank vole is chronic and asymptomatic. It is likely that different coevolutionary histories between PUUV and its hosts could lead to different balances of resistance/tolerance to PUUV infection, at least partly mediated by variable production levels of TNF-alpha. We investigated the hypothesis that bank voles from PUUV endemic areas should exhibit higher levels of tolerance, i.e. lower levels of TNF-alpha production, than bank voles from areas where PUUV prevalence is low. For this purpose, we analysed variations of Tnf-alpha gene expression and promoter sequences among European populations of bank voles. Our results revealed an absence of up-regulation of Tnf-alpha gene expression in PUUV infected bank voles and significant differences in Tnf-alpha gene expression level with regard to PUUV endemicity. These results corroborated the hypothesis of different balances of tolerance/resistance to PUUV. Two single-nucleotide polymorphism genotypes within the Tnf-alpha promoter (-302 GG/GG and -296 A/A) were associated with higher Tnf-alpha gene expression and were more frequent in non-endemic areas. This study emphasized the potential influence of selection acting on TNF-alpha production and mediating a tolerance/resistance balance to PUUV in bank voles. Further investigations, including the role of phenotypic plasticity and parasite communities on Tnf-alpha expression levels, should provide important keys to understand the prevalence of PUUV over Europe.