Education inequality at the neighborhood-level may influence population health and health behavior. We assessed the relations between education inequality and substance use in 59 New York City (NYC) neighborhoods. We used Gini coefficients of education to describe neighborhood education inequality and data from a random-digit-dial phone survey of adult residents of NYC to assess use of substances. Among 1355 respondents (female=56.2%; white=35.7%; mean age=40.4), 23.9% (95% confidence interval [CI]=20.3-27.5) reported smoking, 39.4% (95% CI=35.3-43.4) drinking, and 5.4% (95% CI=3.6-7.3) using marijuana in the previous 30 days. In multilevel models controlling for neighborhood education, neighborhood income inequality, and individual covariates, living in a neighborhood with high education inequality was associated with a greater prevalence of drinking (p=0.02) and of smoking marijuana (p=0.004) but among current drinkers it was associated (p=0.03) with having fewer drinks. The odds of alcohol use (OR=1.70) and marijuana use (OR=3.49) were greater in neighborhoods in the 75th percentile of education Gini compared to neighborhoods in the 25th percentile of education Gini. Statisical interactions suggest that there may be a stronger relation between education inequality and marijuana use in neighborhoods with low mean education than in neighborhoods with higher mean levels of education. These findings, taken together, suggest a complex relation between education inequality and substance use; likelihood of the use of alcohol and marijuana was higher in areas with higher education inequality suggesting potential roles for substance use norms and availability, whereas quantity used among drinkers was higher in areas with low education inequality, suggesting potential roles for both disadvantage and norms.