Antimicrobial resistance (AMR) is a serious health threat, and it has high priority among the European public health agenda. The development and implementation of the National Action Plans (NAP) with a One Health perspective to fight AMR was supported in 2017 by the European Union (EU) through a Joint Action on Antimicrobial Resistance and Healthcare Associated Infections (EU-JAMRAI). The Italian National Institute of Health (Istituto Superiore di Sanità), supported by the University of Udine, and the University of Foggia were among the 44 partners involved. This paper describes the results of EU-JAMRAI relevant to Italy and its impact on national policies Methods: The activities were carried out involving national and international experts who worked in groups, either in virtual or in face-to-face meetings. Country to country visits were organized to assess and compare the national strategies to counteract AMR and to exchange best practices. Qualitative research methods, in particular focus groups (FG) and structured interviews, were carried out to collect information and opinions from the experts. Results: The Italian team of experts from the Ministry of Health and the University of Foggia visited the Netherlands and hosted the Polish expert team in Italy. In two FG, the opinions of stakeholders from different organizations were collected and analyzed to identify critical areas and provide recommendations to ensure implementation of the NAP and effective One Health integration. Attitudes of medical professionals toward antimicrobial stewardship were evaluated in a medium/large Italian hospital. Strengths were identified in the multidisciplinary approach and in the proactive involvement of the hospital management. As for the veterinary sector, Italy was among the 10 EU countries that did not have any national AMR surveillance in place for animal bacterial pathogens. Consequently, a European surveillance system, with the adhesion of Italy, was proposed. Regarding research and innovation to fight AMR and HAI Italy worked with the other European partners to identify national research gaps and opportunities. Recommendations were issued to the authorities to promote research and innovation with a One Health approach. Discussion/Conclusions: The Italian participation in the EU JAMRAI provided experience and examples to the Italian government for the implementation of the NAP and for the planning of the roadmap to fight AMR and helped also in pointing out criticalities of the system. It also supported the promotion of the One Health integrated vision that was included in the updated NAP.