This study examined the association of acculturation in the United States and serum carotenoid levels. The design was a cross-sectional, nationally representative survey of 16,539 participants, 17 years of age and older, from the Third National Health and Nutrition Examination Survey (NHANES III). The main outcome measures were serum levels of ⍺-carotene, β-carotene, β-cryptoxanthin, lutein/zeaxanthin, lycopene, and total carotenoids. Multivariate linear regression was used to model the association of serum carotenoids and country of birth, language of interview, and years in the United States. Adjustments were made for age, sex, years of education, race/ethnicity, body mass index, alcohol use, physical activity, serum cotinine, serum cholesterol, and vitamin/mineral usage. Individuals born in the United States who speak English had the lowest levels of carotenoids, and individuals born in Mexico had the highest levels of carotenoids, with the exception of lycopene. Years of residence in the United States was associated with lower ⍺-carotene (4.18 vs 1.51), β-carotene (20.21 vs 14.87), β-cryptoxanthin (12.51 vs 8.95), lutein/zeaxanthin (25.15 vs 18.03), and total carotenoids (88.79 vs 75.44). Years residence in the United States was positively associated with higher lycopene levels (26.69 vs 32.03). Acculturation in the United States was associated with lower fruit and vegetable intake, as measured by serum carotenoid levels.