Background and objectives: In Yemen, there is currently no specific/published guideline for influenza vaccination. In addition, data on knowledge, attitude, and practice, regarding influenza vaccination among HCWs in Yemen is still not studied yet. This study was conducted to assess the knowledge, attitude, practice, and barriers regarding influenza vaccination among HCWs in Yemen. Methodology: Descriptive cross-sectional KAP study was conducted among HCWS in public and private health care sectors located in Southern-Eastern part of Yemen. The Online Raosoft software was used for sample size calculation. If an adequate knowledge level of 50% among HCWs for a maximum sample size with a margin of error of ±5% and 95% confidence interval, the required sample size was 415 after adding 10% for incomplete responses. The convenience sampling method was used to represent the different geographical locations. A self-structured questionnaire was adapted from various previous studies in Yemen and other surrounding countries regarding influenza vaccines. The questionnaire was modified following an evaluation of technical detail and cultural appropriateness by MOPH&P research Committee and cleared by WHO ethical committee. Descriptive statistics was used to describe the findings of the study regarding seasonal influenza and influenza vaccination. Results: A total of 415 HCWs participated in the study with a 100% response rate. The mean age of participants was 37 years (SD = 10.7) and most of the participants were young adult between 20-35 years (50.6%), out of them were female (37.8%). 64.1% of responders declared poor knowledge toward influenza vaccines. However, most of the responders 373 (89.9%) had never been vaccinated. Lack of knowledge about the disease and the vaccine (205, 49.4%) and lacking trust in the health system (128, 30.8%) were the mean reasons for no vaccination among study participants. A greater proportion of responders cited that, the means source of information about influenza vaccination is HCWs (310=74.7%). A 62.2% of participants believed that Influenza vaccine is important and should be taken yearly. The majority of the participants declared their positive acceptance 279= 67 %. Conclusion: This study highlights the fact that generally acceptance of influenza vaccination of healthcare workers in Yemen, and the need to increase efforts and awareness in the community including health care workers regarding influenza illness and vaccination to reduce seasonal influenza morbidity and mortality and consequently the economic and social burden. The findings of the current study could help devise an immunization policy for HCWs in Yemen.