OBJECTIVES To describe compliance with the central line (CL) insertion bundle overall and with individual bundle elements in US adult intensive care units (ICUs) and to determine the relationship between bundle compliance and central line-associated bloodstream infection (CLABSI) rates. DESIGN Cross-sectional study. PARTICIPANTS National sample of adult ICUs participating in National Healthcare Safety Network (NHSN) surveillance. METHODS Hospitals were surveyed to determine compliance with CL insertion bundle elements in ICUs. Corresponding NHSN ICU CLABSI rates were obtained. Multivariate Poisson regression models were used to assess associations between CL bundle compliance and CLABSI rates, controlling for hospital and ICU characteristics. RESULTS A total of 984 adult ICUs in 632 hospitals were included. Most ICUs had CL bundle policies, but only 69% reported excellent compliance (≥95%) with at least 1 element. Lower CLABSI rates were associated with compliance with just 1 element (incidence rate ratio [IRR] 0.77; 95% confidence interval [CI], 0.64-0.92); however, ≥95% compliance with all 5 elements was associated with the greatest reduction (IRR, 0.67; 95% CI, 0.59-0.77). There was no association between CLABSI rates and simply having a written CL bundle policy nor with bundle compliance <75%. Additionally, better-resourced infection prevention departments were associated with lower CLABSI rates. CONCLUSIONS Our findings demonstrate the impact of transferring infection prevention interventions to the real-world setting. Compliance with the entire bundle was most effective, although excellent compliance with even 1 bundle element was associated with lower CLABSI rates. The variability in compliance across ICUs suggests that, at the national level, there is still room for improvement in CLABSI reduction. Infect Control Hosp Epidemiol 2016;37:805-810.