Total circulating 25-hydroxyvitamin D [25(OH)D)] has been associated with lower risk of colorectal cancer (CRC). The physiologic mechanism, however, may be more directly related to the free or bioavailable fraction of 25(OH)D, which is influenced by levels of vitamin D binding protein (VDBP). We assessed the association of prediagnosis total, free and bioavailable 25(OH)D and VDBP with CRC risk among predominantly white women in the Nurses’ Health Study (NHS) who provided a blood specimen in 1989–1990. We documented 378 cases of CRC through 2011 and matched them to 689 controls according to age and time of blood draw. We genotyped two common polymorphisms in the gene coding VDBP, and calculated free and bioavailable 25(OH)D levels based on total 25(OH)D, VDBP, albumin, and their estimated genotype-specific binding affinities. Total 25(OH)D was associated with lower CRC risk (P for trend=0.01). Compared to women in the lowest quintile of total 25(OH)D, those in the highest quintile had an multivariable-adjusted odds ratio (OR) for CRC of 0.54 (95% CI, 0.33–0.87). Comparing extreme quintiles, we did not find any significant association with risk of CRC for VDBP (OR, 0.98; 95% CI, 0.65–1.47); free 25(OH)D (OR, 0.71; 95% CI, 0.46–1.10); or bioavailable 25(OH)D (OR, 0.92; 95% CI, 0.60–1.42). In conclusion, prediagnosis levels of total, but not free or bioavailable 25(OH)D, were associated with lower CRC risk. Although our findings support an inverse association of vitamin D with CRC, this association does not appear to be due to the unbound or bioavailable fraction of circulating vitamin D.