Background: Cardiovascular (CV) implications of long-term gender affirming hormonal treatment (GAHT) in transgender individuals still remain largely unknown. Aim: To evaluate changes in the 30-year Framingham cardiovascular disease (CVD) risk in a large cohort of transgender individuals after the start of GAHT. Methods: In a multicenter prospective study, a consecutive series of 309 participants (165 transmen and 144 transwomen) was evaluated during a 2-year follow-up. Prospectively, after the start of GAHT a physical examination was performed and blood samples were drawn. CVD risk was calculated for each person, according to the Framingham 30-year CVD risk estimate. Main Outcome Measure: Changes in CV risk factors and 30-year Framingham CVD risk during GAHT. Clinical Implications: In transmen testosterone-induced lipid profile alterations may have a clinical relevance on the individual long-term CVD risk. Strengths & Limitations: The strength of the present study is the possibility to predict long-term CV outcomes in transgender individuals receiving GAHT based on a short observation; whereas the main limitation is that CVD risk prospective changes mainly represent the expression of risk factors changes during GAHT. Results: In transwomen a significant decrease in triglycerides, total cholesterol and LDL-cholesterol was observed during the 2-year follow-up (P <.05), whereas unfavorable lipid changes – such as increased total cholesterol, triglycerides, and LDL cholesterol levels and decreased HDL cholesterol levels (P <.05)- occurred after the start of GAHT in transmen. These changes in risk factors led to an increase in the risk of general and hard CVD events based on lipid profile over time in transmen (P =.001 and P =.005, respectively). No significant changes in general and hard CVD risk based on lipid profile were observed in transwomen over time. Conclusions: Our findings confirmed the unfavorable lipid changes in transmen after the start of GAHT even during a longer follow-up, empathizing the potential clinical impact of these modifications on individual long-term CVD risk. Cocchetti C, Castellini G, Iacuaniello D, et al. Does Gender-Affirming Hormonal Treatment Affect 30-Year Cardiovascular Risk in Transgender Persons? A Two-Year Prospective European Study (ENIGI). J Sex Med 2021;xxx:xxx–xxx.