Background: Vitamin D is essential for health and its shortage exacerbate overall mortality. Health care workers (HCWs) need to educate on its uses and sources although studies indicate their low level of practice. 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 ecologies covering lowland, midland and highland districts. A total of 405 health care workers with different levels 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 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: 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 on adult vitamin D deficiency. Besides, HCWs residential ecology, clinical position, knowledge and attitude 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.