Objectives: Periodical assessments of population susceptibility to polioviruses (PV) is essential for evaluating population protection and planning appropriate vaccination strategies. The aim of the current work was to assess serological protective titers against all three polioviruses in the general population of Florence. Methods: A convenience sample of 328 sera, collected in 2009 in Florence (Central Italy) was analyzed. Samples were considered protective if neutralizing antibodies were detected at dilutions ≥1:8, according to the WHO protocols. Results: The immune coverage was 75.3%, 69.2% and 46% for PV1, PV2 and PV3, respectively. The protective titers of neutralizing antibodies were generally higher in children up to 14 years of age, with 74.4% (PV1), 75.6% (PV2) and 56.7% (PV3) of seroprevalence. From the age of 11 years, most of the study subjects were seronegative for PV3. Conclusions: In a polio-free country with strong migration pressures, such as Italy, our results bring clear support to the recent recommendation of Italian health authorities to introduce a fifth dose of IPV vaccine in adolescence all over the country.