The Natura 2000 network is the most important conservation effort being implemented in Europe. Nevertheless, no comprehensive and systematic region—or nationwide evaluation of the effectiveness of the network has been conducted. We used habitat suitability models and extent of occurrence of 468 species of vertebrates to evaluate the contribution of the Natura 2000 network to biodiversity conservation in Italy. We also estimated the population size of 101 species inside the Natura 2000 network to assess its capacity to maintain or improve the population status of listed species. In general the Italian Natura 2000 did not seem to integrate existing protected areas well. The Natura 2000 network increased from 11% to 20% the area devoted to conservation in Italy and the coverage provided to areas with high biodiversity. Nevertheless, some areas with high numbers of species were devoid of conservation areas, and more than 50% of the highly irreplaceable areas were not considered in the system. Moreover, the Natura 2000 network cannot maintain 44-80% (depending on the taxa considered) of the species in a “favorable conservation status” under World Conservation Union Red List criteria. The Natura 2000 network is probably stronger than the results of our analyses suggest. The system is based on a site-specific expert-based strategy and is driven by direct and detailed knowledge of local diversity. Nevertheless, if Natura 2000 is taken to represent the final point of all the EU conservation policies, it will inevitably fail. Its role in conservation could be enhanced by integrating the Natura 2000 system into a more general strategy that considers natural processes and the ecological and evolutionary mechanisms underlying these processes.