Elevated winter losses of managed honeybee colonies are a major concern, but the underlying mechanisms remain controversial. Among the suspects are the parasitic mite Varroa destructor, the microsporidian Nosema ceranae, and associated viruses. Here we hypothesize that pathogens reduce the life expectancy of winter bees, thereby constituting a proximate mechanism for colony losses. A monitoring of colonies was performed over 6 months in Switzerland from summer 2007 to winter 2007/2008. Individual dead workers were collected daily and quantitatively analyzed for deformed wing virus (DWV), acute bee paralysis virus (ABPV), N. ceranae, and expression levels of the vitellogenin gene as a biomarker for honeybee longevity. Workers from colonies that failed to survive winter had a reduced life span beginning in late fall, were more likely to be infected with DWV, and had higher DWV loads. Colony levels of infection with the parasitic mite Varroa destructor and individual infections with DWV were also associated with reduced honeybee life expectancy. In sharp contrast, the level of N. ceranae infection was not correlated with longevity. In addition, vitellogenin gene expression was significantly positively correlated with ABPV and N. ceranae loads. The findings strongly suggest that V. destructor and DWV (but neither N. ceranae nor ABPV) reduce the life span of winter bees, thereby constituting a parsimonious possible mechanism for honeybee colony losses.