Background: The aim of this study is to assess the acceptability of HIV pre-exposure prophylaxis (PrEP) among individuals eligible for prophylaxis. Setting: Retrospective study of individuals receiving their first HIV test at the Infectious Diseases Unit of IRCCS San Raffaele Scientific Institute, Milan, Italy. Methods: People i) who received their first HIV test (baseline date) between January 2018 (availability of PrEP in Italy) and December 2023 (data lock), ii) with an indication for PrEP based on condomless sex were included; those already on PrEP were excluded. Individuals with a negative HIV test result were offered PrEP counselling and prescription according to guidelines. Characteristics of people who accepted or declined HIV PrEP were compared using Mann-Whitney or Chi-square tests, as appropriate. Results: A total of 2627 people were tested for HIV: 175 (6.6%) were diagnosed with HIV. Of the 2452 people with negative HIV test results, 2165 (88.3%) were men; median age was 34.3 years (interquartile range, IQR=28.7-42.4). Sexually transmitted infections (STIs) were detected in 478/2452 (19.5%), most commonly gonorrhoea (206/2452, 8.4%). Overall, 982/2452 (40.0%) people accepted PrEP, including only three women. Non-acceptance was higher among women (p<0.001) and non-Caucasians (p<0.001). Of those who did not accept PrEP, 260 (17.7%) were diagnosed with at least one STI at the time of HIV testing. Conclusion: Low PrEP acceptance was observed, particularly among women and people of non-Caucasian origin. Non-acceptance of PrEP was also observed among individuals diagnosed with an STI.