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Objectives: Influenza vaccination is important for healthcare workers in order to prevent both the illness itself and transmission to patients. Previous studies in Saudi Arabia have revealed low influenza vaccine coverage among healthcare workers due to misconceptions. This study aimed to assess knowledge, attitudes, and practices regarding influenza vaccination among healthcare workers during 2021, addressing the current data gap. Methods: A cross-sectional study was conducted, including 1273 healthcare workers from the Ministry of Health in Saudi Arabia. A self-administered questionnaire was distributed to participants via email. Results: Most participants had an appropriate extent of knowledge, with 37.1% having a high level and 26.6% having a moderate level. Positive attitudes toward the influenza vaccine were observed in 41.2% of participants, and 80.2% demonstrated good vaccine practices. However, the vaccine coverage was 50.8% in the past 12 months. Factors associated with vaccine uptake included previous vaccination, workplace availability, awareness of guidelines, engagement in training programs, type of workplace settings, and having positive attitudes toward the vaccine. The most common reason for not getting vaccinated was the perception of being at low risk, making vaccination unnecessary. Conclusions: Participants exhibited positive knowledge, attitudes, and practices regarding influenza vaccination. However, the observed vaccine uptake rate fell below the recommended coverage rate, indicating the presence of a knowledge-behavior gap. Targeted interventions are recommended to improve vaccination uptake among healthcare workers in Saudi Arabia.