Background. With the aim to inform potential SHE programs in Italian schools, we run a survey on 4,552 freshmen of four different Italian universities (Padua, Palermo, Bergamo and Milan) to investigate their level of SHK on STI prevention and associated factors. Methods. The survey was conducted with an anonymous self-reported paper questionnaire administered during teaching lectures to university freshmen of four Italian universities and different undergraduate courses. Results. The final number of freshmen surveyed was 4,552 (97.9% response rate), 1,842 (40.5%) from the university of Padua; 1,743 (38.3%) from the university of Palermo; 667 (14.7%) from the university of Bergamo and 300 (6.6%) from the university of Milan. The mean age of respondents was 21.4 2.2 years and most of them (70.3%) were females. An overall low STI knowledge among sample students was found. Only 28% respondents knew the most effective contraceptives to prevent STI (i.e. condom and sexual abstinence), with a slightly higher prevalence of correct answers among females (31.3%) than males (25.8%). Students with history of STIs reported referring mostly to their GP (38.1%), rather than discussing the problem with a family member (15.1%), booking an appointment with a consultant gynecologist (13.1%), discussing with their partner (13.1%) or with an experienced friend (12.5%). At multivariable analysis significantly lower levels of STI knowledge was found among males, students of the university of Palermo, those who completed a vocational secondary school education and non-Italian nationals. Categories with significantly less SHK on the most effective contraceptives to prevent STIs included males, students of the university of Palermo, students of educational sciences, students of economics/political sciences, those of foreign nationality, those with two siblings and those whose fathers had a lower educational level. Conclusions. The above findings provided indirect information on the standard of SHE delivered in Italian schools and call for the development of tailored multi-agency SHE programs involving several actors. In addition to school-based SHE programs, primary care services dedicated to sexual health should be strengthened in Italy.