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Vitamin D deficiency is associated with susceptibility to tuberculosis (TB) in HIV-uninfected people in Europe, but it is not known whether such an association exists among HIV-infected people in subtropical Africa. We conducted a cross-sectional study to determine whether vitamin D deficiency was associated with susceptibility to active TB in HIV-uninfected (n = 196) and HIV-infected (n = 174) black Africans in Cape Town, South Africa. We also investigated whether there was evidence of seasonal variation in vitamin D status and TB notifications in this setting over an 8-y period. Vitamin D deficiency (serum 25-hydroxyvitamin D [25(OH)D] <50 nmol/L) was present in 232 (62.7%) of 370 participants and was associated with active TB in both HIV-uninfected (odds ratio = 5.2, 95% confidence interval: 2.8-9.7; P < 0.001) and HIV-infected (odds ratio = 5.6, 95% confidence interval: 2.7-11.6; P < 0.001) people. Vitamin D status varied according to season: The mean serum 25(OH)D concentration was highest in January through March and lowest in July through September (56.8 vs. 30.7 nmol/L, respectively; P < 0.001). Reciprocal seasonal variation in TB notifications was observed: The mean number of TB notifications per quarter for Cape Town in 2003 to 2010 was lowest in April through June and highest in October through December (4,222 vs. 5,080; P < 0.001). Vitamin D deficiency is highly prevalent among black Africans in Cape Town and is associated with susceptibility to active TB both in the presence and absence of HIV infection. Reciprocal seasonal variation in serum 25(OH)D concentration and TB notifications suggests that seasonal variations in vitamin D status and TB incidence in this setting are causally related. [ABSTRACT FROM AUTHOR]