<p><b>BACKGROUND: </b>Adherence to the American Cancer Society (ACS) guidelines for cancer prevention is associated with a lower risk of cancer and mortality. The role of neighborhood segregation on adherence to the guidelines among Hispanic/Latino adults is relatively unexplored.</p><p><b>MATERIALS AND METHODS: </b>The Hispanic Community Health Study/Study of Latinos is a community-based prospective cohort of 16,462 Hispanic/Latino adults, ages 18-74 years enrolled in 2008-2011 from the Bronx, Chicago, Miami and San Diego. Dimensions of neighborhood segregation were measured using 2010 United States' census tracts:-evenness (the physical separation of a group), exposure (the propensity for contact between groups), and their joint effect (hypersegregation). ACS guideline adherence levels - low, moderate, high - were created from accelerometry-measured physical activity, dietary intake, alcohol intake, and body mass index. Weighted multinominal logistic regressions estimated relative risk ratios (RRR) and 95% confidence intervals (CI) for guideline adherence levels and its components.</p><p><b>RESULTS: </b>Hispanic/Latino adults were classified as low (13.7%), moderate (58.8%) or highly (27.5%) adherent to ACS guidelines. We found no evidence of an association between segregation and overall guideline adherence. Exposure segregation associated with lower likelihood of moderate adherence to alcohol recommendations (RRR:0.86, 95%CI:0.75-0.98) but higher likelihood for diet recommendations (RRR:1.07, 95%CI:1.01-1.14). Evenness segregation associated with lower likelihood of high adherence to the physical activity recommendations (RRR:0.73, 95%CI:0.57-0.94). Hypersegregation was associated with individual guideline components.</p><p><b>CONCLUSION: </b>We found evidence of a cross-sectional relationship between neighborhood segregation and ACS cancer prevention guideline components, but not with overall ACS guideline adherence.</p>