The surveillance of latent tuberculosis infection (LTBI) in both healthcare workers and healthcare students is considered fundamental for tuberculosis (TB) prevention. The aim of the present study was to estimate LTBI prevalence and evaluate potential risk-factors associated with this condition in a large cohort of medical students in Italy. In a cross-sectional study, performed between March and December 2012, 1511 eligible subjects attending the Medical School of the University of Genoa, trained at the IRCCS San Martino-IST Teaching Hospital of Genoa, were actively called to undergo the tuberculin skin test (TST). All the TST positive cases were confirmed with an interferon-gamma release assay (IGRA). A standardized questionnaire was collected for multivariate risk analysis. A total of 1302 (86.2%) students underwent TST testing and completed the questionnaire. Eleven subjects (0.8%) resulted TST positive and LTBI diagnosis was confirmed in 2 (0.1%) cases. Professional exposure to active TB patients (OR 21.7, 95% CI 2.9-160.2; P value 0.003) and previous BCG immunization (OR 28.3, 95% CI 3.0-265.1; P value 0.003) are independently associated with TST positivity. Despite the low prevalence of LTBI among Italian medical students, an occupational risk of TB infection still exists in countries with low circulation of Mycobacterium tuberculosis.