Abstract Vitamin D has been hypothesized to protect against cancer. We followed 16,819 participants in NHANES III (Third National Health and Nutritional Examination Survey) from 1988 to 2006, expanding on an earlier NHANES III study (1988–2000). Using Cox proportional hazards regression models, we examined risk related to baseline serum 25-hydroxyvitamin D [25(OH)D] for total cancer mortality, in both sexes, and by racial/ethnic groups, as well as for site-specific cancers. Because serum was collected in the south in cooler months and in the north in warmer months, we examined associations by collection season (“summer/higher latitude” and “winter/lower latitude”). We identified 884 cancer deaths during 225,212 person-years. Overall cancer mortality risks were unrelated to baseline 25(OH)D status in both season/latitude groups, and in non-Hispanic whites, non-Hispanic blacks, and Mexican-Americans. In men, risks were elevated at higher levels {e.g., for ≥100 nmol/L, relative risk (RR) = 1.85 [95% confidence interval (CI), 1.02–3.35] compared with <37.5 nmol/L}. Although risks were unrelated to 25(OH)D in all women combined, risks significantly decreased with increasing 25(OH)D in the summer/higher latitude group [for ≥100 nmol/L, RR = 0.52 (95% CI, 0.25–1.15) compared with <37.5 nmol/L; Ptrend = 0.03, based on continuous values]. We also observed a suggestion of an inverse association with colorectal cancer mortality (Ptrend = 0.09) and a positive association with lung cancer mortality among males (Ptrend = 0.03). Our results do not support the hypothesis that 25(OH)D is associated with reduced cancer mortality. Although cancer mortality in females was inversely associated with 25(OH)D in the summer/higher latitude group, cancer mortality at some sites was increased among men with higher 25(OH)D. These findings argue for caution before increasing 25(OH)D levels to prevent cancer. Cancer Res; 70(21); 8587–97. ©2010 AACR.