We examined cross-sectional associations among neighborhood- and individual-level factors related to a healthful lifestyle and dietary intake, physical activity (PA), and support for obesity prevention polices in rural eastern North Carolina adults. We examined perceived neighborhood barriers to a healthful lifestyle, and associations between neighborhood barriers to healthy eating and PA, participants' support for seven obesity prevention policies, and dependent variables of self-reported dietary and PA behaviors, and measured body mass index (BMI) (n = 366 study participants). We then used participants' residential addresses and Geographic Information Systems (GIS) software to assess neighborhood-level factors related to access to healthy food and PA opportunities. Correlational analyses and adjusted linear regression models were used to examine associations between neighborhood-level factors related to a healthful lifestyle and dietary and PA behaviors, BMI, and obesity prevention policy support. The most commonly reported neighborhood barriers (from a list of 18 potential barriers) perceived by participants included: not enough bicycle lanes and sidewalks, not enough affordable exercise places, too much crime, and no place to buy a quick, healthy meal to go. Higher diet quality was inversely related to perceived and GIS-assessed neighborhood nutrition barriers. There were no significant associations between neighborhood barriers and PA. More perceived neighborhood barriers were positively associated with BMI. Support for obesity prevention policy change was positively associated with perceptions of more neighborhood barriers. Neighborhood factors that promote a healthful lifestyle were associated with higher diet quality and lower BMI. Individuals who perceived more neighborhood-level barriers to healthy eating and PA usually supported policies to address those barriers. Future studies should examine mechanisms to garner such support for health-promoting neighborhood changes.