HLA molecules possess polymorphic and antigenic properties making them susceptible to targeting by anti-HLA antibodies due to allo-immunization. This immune response can arise from various circumstances such as transfusions, transplants, and pregnancies. The purpose of our study was to evaluate the effects of pregnancy on the development of anti-HLA antibodies in women awaiting kidney transplantation. We conducted a retrospective analysis encompassing a total of eighty-five women who were tested for anti-HLA immunization using Labscreen Single Antigen tests as part of their pre-transplant assessment. Among the thirty-seven women who had been pregnant, thirty-two (86%; 95%CI = [74%-98%]) tested positive for anti-HLA antibodies. Specifically, twenty-nine (78%; 95%CI = [64%-92%]) had antibodies against HLA class I, and twenty-eight (76%; 95%CI = [61%-90%]) against HLA class II. Regarding the reactivity of these antibodies to the general population, we found that the median PRA was 47% for HLA class I antibodies and 28% for HLA class II antibodies. Furthermore, we observed a positive correlation between the number of pregnancies and the PRA, with a statistical measure (R²) of 0.35 (p = 2.5e-9) for HLA class I antibodies and 0.29 (p = 9.2e-8) for HLA class II antibodies. Multivariate analysis demonstrated that the presence of at least one pregnancy significantly increased the likelihood of anti-HLA allo-immunization, regardless of age and number of pregnancies (p < 0.01; OR = 57.9; 95%CI = [8.61 - 643]). In conclusion, our study demonstrates that a single pregnancy is sufficient to induce the development of anti-HLA antibodies. Additionally, the number of pregnancies impacts the diversity of the antibody response. These findings underscore the importance of considering pregnancy as a factor in assessing the risk of anti-HLA allo-immunization and its potential impact on transplant outcomes.