Carotenoids are readily absorbed from the diet and distributed in blood leukocyte subcellular organelles. Bixin, a potent bioactive found in the seed of the Annatto plant, , possesses antioxidant and anti-inflammatory properties. The purpose of this study was to determine the uptake of bixin by plasma, lipoproteins, and leukocytes in domestic dogs and to examine immunoprotective properties. To determine uptake kinetics, female Beagle dogs (2 yr; 9.1 ± 0.1 kg BW) were first fed a single dose by oral gavage of 0, 5, 10, 20, or 40 mg bixin, with blood collected at 0 to 16 h after administration ( = 6/treatment), and then fed daily with 0, 5, 10, 20, or 40 mg bixin/d, with blood collected at 0, 1, 2, 4, 6, 10, and 14 d. In a consecutive experiment, cell-mediated and humoral responses as well as oxidative biomarkers were measured following 16 wk of dietary supplementation with 0, 5, 10, or 20 mg bixin/d. Maximal absorption in plasma occurred by 0.5 h with an elimination half-life of 2.6 to 3.3 h after a single dose of bixin. Steady-state plasma concentrations were 0.053 μ after 14 d of 40 mg bixin/d. The majority of subcellular bixin was found in the leukocyte mitochondria and was associated with the high-density lipoprotein and low-density lipoprotein fractions of lipoproteins. Specific (vaccine) response increased ( < 0.05) but nonspecific mitogen response was unchanged after 12 wk of dietary bixin, as assessed by a delayed-type hypersensitivity assay. Both B cell plasma leukocyte subpopulations at 6 and 16 wk and IgG plasma concentration at 12 wk in the 10-mg treatment group increased ( < 0.05), although IgM production and other cell populations were unaffected. In addition, 8-oxo-2'-deoxyguanosine (8-OHdG), a DNA damage biomarker, was substantially reduced ( < 0.05) in all treatment groups by wk 16, and C-reactive protein (CRP) was suppressed at wk 12 ( < 0.05). Dietary supplementation with bixin showed no changes in lymphoproliferation in response to in vitro mitogenic challenge and had no effect in enhancing natural killer cell activity. In conclusion, bixin was readily absorbed in a dose-dependent manner in blood following oral administration and was then taken up by leukocytes, where it was primarily distributed to mitochondria but in other subcellular organelles as well. Bixin also appeared to stimulate immune response, as seen with cell-mediated responses, and exerted anti-inflammatory (reduced CRP) as well as antioxidative (reduced 8-OHdG) effects in dogs.