Background: Vitamin D is an immunomodulator, and its deficiency is associated with Tuberculosis (TB) infection. Bronchoalveolar lavage fluid (BALF) is a rich milieu of macrophages that form the first line of defense against invading TB bacilli. As there is an increased prevalence of vitamin D deficiency in TB and human immunodeficiency virus-1 (HIV-1) subjects, we intend exploring the possibility of a localized deficiency of vitamin D metabolites in BALF of these patients. Objective: The primary objective was to assess the level of 25D 3 in serum and BALF of subjects and look for a significant difference among patients and controls. The secondary objective was to find a correlation between serum and BALF 25D 3 levels. Methods: We performed a cross-sectional study with subjects divided into four groups: Controls (group 1), HIV positive without active TB (group 2), active TB without HIV (group 3), and HIV-TB coinfection (group 4). BALF and serum 25D 3 levels were compared between the groups. Results: Among the 149 (an immunomodulator) successive subjects enrolled, there were 40 subjects in group 1 (HIV-TB-), 48 in group 2 (HIV+TB-), 37 in group 3 (HIV-TB+), and 24 in group 4 (HIV+TB+). Females constituted 31.6% of the study subjects. In groups 3 and 4, there were significantly lower serum 25D 3 levels compared to group 1 (p-value group 3: 0.002; group 4: 0.012). In groups 2, 3, and 4, there were significantly lower BALF 25D 3 levels compared to group 1 (p-value group 2: 0.000; group 3: 0.000; group 4: 0.001). There was a significant correlation between serum and BALF 25D 3 levels (Spearman’s rank correlation coefficient 0.318, p-value = 0.0001). Conclusion: Lower levels of serum and BALF 25D 3 were observed in HIV, TB, and HIV-TB coinfected patients. Localized deficiency of vitamin D metabolites might be associated with increased vulnerability to TB infection.