Background: Syphilis infection does not confer definitive and protective immunity against reinfection, and crucial aspects of repeated episodes of syphilis are far from being understood, especially among people living with HIV (PLWH). Methods: In order to explore the burden of syphilis in a large cohort of HIV-negative patients and PLWH, this retrospective study describes the demographics, clinical presentation and treatment outcome of patients with syphilis treated at our clinic from 2013 to 2021. Results: Within the study period, 1859 syphilis episodes (827, 44.5% first infections and 1032, 55.5% reinfections) were recorded. A total of 663 patients, of whom 347 (52%) had PLWH, were considered. Syphilis was mostly diagnosed in males (77%) and European (79%) patients. More than half of syphilis episodes were recorded during the late latent stage (64%) or during follow-up/screening visits for other diseases, while symptomatic stages led to a diagnosis in almost half of HIV-negative patients (p < 0.001). PLWH with syphilis infection were predominantly homo/bisexual (p < 0.001). A significantly higher rate of syphilis reinfection was observed in PLWH, who also demonstrated a higher range of subsequent episodes. The serofast state was found to be similar at the 6- and 12-month follow-up visits. The multivariate analysis carried out in the HIV-positive group showed that an RPR titre >1:16 was an independent predictor for serological non-response. Conclusions: Syphilis reinfections are predominantly diagnosed in HIV-positive MSM. The high rate of asymptomatic presentation among PLWH supports the role of periodical syphilis screening. In PLWH, the only baseline factor associated with an increased risk of non-response was an RPR titre >1:16, while assessment at 12 months after treatment increased the possibility of detecting a serological response, indicating that PLWH have a slower serological response to treatment.