Background: Early care and education (ECE) is an important setting for influencing young children's dietary intake. There are several factors associated with barriers to healthy eating in ECE, and recent evidence suggests that participation in the Child and Adult Care Food Program (CACFP), the primary national food assistance program in ECE, may be associated with fewer barriers to serving healthier foods. However, no prior studies have examined differences between CACFP participants and non-participants across a large, multi-state sample. This is the first study to examine the association between CACFP participation and barriers to serving healthier foods in ECE using a random sample of directors from two regions across the country. Methods: We conducted a cross-sectional survey among a random sample of child care center directors from four states (Massachusetts, North Carolina, Rhode Island, and South Carolina). We conducted logistic and Poisson regression to calculate the odds and incidence rate ratios of reporting 1) no barriers, 2) specific barriers (e.g., cost), and 3) the total number of barriers, by CACFP status, adjusting for covariates of interest. Results: We received 713 surveys (36% response rate). About half (55%) of centers participated in CACFP. The most prevalent reported barriers to serving healthier foods were cost (42%) and children's food preferences (19%). Directors from CACFP centers were twice as likely to report no barriers, compared to directors from non-CACFP centers (OR 2.03; 95% CI [1.36, 3.04]; p 0.01). Directors from CACFP centers were less likely to report cost as a barrier (OR = 0.46; 95% [CI 0.31, 0.67]; p 0.001), and reported fewer barriers overall (IRR = 0.77; 95% CI [0.64, 0.92]; p 0.01), compared to directors from non-CACFP centers. Conclusions: CACFP directors reported fewer barriers to serving healthier foods in child care centers. Still, cost and children's food preferences are persistent barriers to serving healthier foods in ECE. Future research should evaluate characteristics of CACFP participation that may alleviate these barriers, and whether barriers emerge or persist following 2017 rule changes to CACFP nutrition standards. (author abstract)