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Background: Late HIV diagnosis remains one of the challenges in combating the epidemic. Primary care providers play an important role in screening for HIV infection. Our study aims to evaluate the relationship between knowledge and barriers to HIV testing and screening outcomes. The impact of an education program for primary care providers, towards improving HIV testing and late diagnosis rates, is also assessed. Methods: A self-administered questionnaire that was developed within the framework of the European project OptTEST was used to examine HIV knowledge and barriers to HIV testing scores before and after being involved in an HIV education program. A quasi-experimental design with pre- and post-intervention measures was performed to investigate its impact. We performed multivariable logistic regression analysis to assess the relationship between variables for the HIV testing offer. Results: A total of 20 primary care centers and 454 primary care staff were included. Baseline OptTEST results showed that more knowledgeable staff offered an HIV test more frequently (OR 1.07; CI 95% 1.01–1.13; p = 0.027) and had lower barrier scores (OR 0.89; CI 95% 0.77–0.95; p = 0.005). Nurses had lower scores in knowledge-related items (OR 0.28; CI 95% 0.17–0.46; p<0.001), but higher scores in barrier-related items than physicians (OR 3.28; CI 95% 2.01–5.46; p<0.001). Specific centers with more knowledgeable staff members had a significant association with a greater level of new HIV diagnosis rates (OR 1.61; CI 95% 1.04–2.49; p = 0.032). After the intervention, we found that 12 out of 14 individual questions showed improved scores. In the 6 months after the training program, we similarly found a higher HIV testing rate (OR 1.19; CI 1.02–1.42; p = 0.036). Conclusions: This study highlights the association between knowledge and barriers to HIV testing, including HIV testing rates. It shows that it is possible to modify knowledge and reduce perceived barriers through educational programs, subsequently improving HIV screening outcomes. [ABSTRACT FROM AUTHOR]