Vitamin D is a fat-soluble vitamin that increases immunity against tuberculosis (TB), decreases the re-activation of latent TB and reduces the severity of active TB disease. Epidemiological studies on the prevalence of vitamin D deficiency and its association with TB have shown inconsistent results in different countries. This study aimed to determine the prevalence of vitamin D deficiency and its association with TB in Northwest Ethiopia. A case–control study was conducted among smear positive pulmonary TB patients and their household s without symptoms suggestive of TB. Study participants were recruited at 11 TB diagnostic health facilities in North and South Gondar zones of Amhara region between May 2013 and April 2015. Vitamin D deficiency is highly prevalent among TB patients and non-TB controls in Ethiopia, where there is year-round abundant sunshine. Study participants with TB, females, older age groups, and urban residents had significantly higher prevalence of vitamin D deficiency. These findings warrant further studies to investigate the role of vitamin D supplementation in the prevention and treatment of TB in high TB burden countries like Ethiopia.