Background: Childhood obesity is a national epidemic that disproportionately affects Hispanic children. Evidence suggests that increased acculturation among this population adversely affects diet and other healthy lifestyle characteristics, leading to higher rates of overweight and obesity. Healthy lifestyle characteristics must be understood in order to prevent or decrease overweight and obesity among Hispanic children. Methods: Using the School Physical Activity and Nutrition (SPAN) study, we examined cross-sectional data on healthy lifestyle characteristics collected in Texas public schools from Hispanic fourth-grade children in 2004-2005. We calculated adjusted odds ratios and associated confidence intervals using multivariate logistic regression analyses to analyze the association between acculturation and healthy lifestyle characteristics among Spanish-speaking Hispanic children compared to English-speaking Hispanic children. Results: Spanish-speaking Hispanic boys consumed more milk and fruit than English-speaking Hispanic boys (milk: adjusted odds ratio [AOR]: 1.7, p = 0.02; fruit: AOR: 2.5, p = 0.0001). The likelihood that Spanish-speaking Hispanic boys and girls did not know that there is a relationship between overweight and health problems were 2 times greater (boys: AOR: 1.7, p = 0.03; girls: AOR: 2.2, p = 0.006) than their English-speaking Hispanic counterparts. Likelihood of weight loss attempts was greater among Spanish-speaking Hispanic boys than English-speaking Hispanic boys (AOR: 1.9, p = 0.04). Conclusions: Results are mixed. Lower levels of acculturation appear to be associated with both positive and negative healthy lifestyle characteristics, depending on sex. These findings have important implications for school health policies and programs and should be distributed to school administrators. (Contains 3 tables.)