Background: Vitamin D is very essential for health and its insufficiency is strongly associated with increased overall mortality. Health care workers (HCWs) need to educate on its uses and possible sources although studies show that their knowledge, attitude and practice is low; few studies are available in Ethiopia. The main aim of this study is therefore to assess the knowledge, attitude and practice of measuring adult vitamin D status, diagnosis of deficiency and managing health consequences among HCWs in Ethiopia. Methods: This study was conducted in three districts covering lowland, midland and highland ecologies. A total of 405 health extension workers, nurses, health officers, general practitioners and specialists were interviewed. Tablets were used for data collection to archive in a cloud server. Data were exported to Stata version 14 software for cleaning and analysis. Rates were computed and Chi-square test was used to compare differences between the two groups. Binary logistic regression was used to measure the strength, direction and significance of association between different covariates and the practice of HCWs.Result: The level of good knowledge, positive attitude and good practice in measuring adult vitamin D status, diagnosis of deficiency and managing health consequences among HCWs was 210(51.8%), 261(63.5%) and 195(47.4%) respectively. The odds of good practice in the provision of adult vitamin D service were AOR=6.87: 95% CI (3.57, 13.21) and AOR= 2.20: 95% CI (1.23, 3.92) times higher among HCWs in Addis Ababa and highlands compared with those working in lowlands. Good practice among clinicians was AOR=4.26: 95% CI (1.48, 12.25) times higher compared with those working in leadership positions. The likelihood was AOR=1.96: 95% CI (1.19, 3.23) times higher among those with good knowledge compared with those with poor knowledge. Besides, good practice in adult vitamin D service provision was AOR=2.30: 95% CI (1.40, 3.78) times higher among those with positive attitude compared with those who had negative attitude.Conclusions: Only a little over half of HCWs have good knowledge and close to two-third of them has positive attitude while less than half of them have good practice of measuring adult vitamin D status, diagnosis and managing its health consequences practice. Besides, HCWs residential ecology, clinical position, good knowledge and positive associated is associated with good practice on adult vitamin D. It is essential to provide rigorous and continues training for HCWs focusing on their deployment ecology.