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Background: Culturally valid measures of depression for Spanish-speaking Hispanic women are important for developing and implementing effective interventions to reduce health disparities. The Center for Epidemiological Studies-Depression Scale (CES-D) is a widely used measure of depression. Differential item functioning has been studied using language preference as a proxy for acculturation, but it is unknown if the results were due to acculturation or the language of administration., Objective: The aim of this study was to evaluate the relationship of acculturation, defined with a dimensional measure, to Spanish CES-D item responses., Methods: Spanish-speaking Hispanic women (n = 504) were recruited for a randomized controlled trial of Salud, Educación, Prevención y Autocuidado (Health, Education, Prevention, and Self-Care). Acculturation, an important dimension of variation within the diverse U.S. Hispanic community, was defined by high or low scores on the Americanism subscale of the Bidimensional Acculturation Scale. Differential item functioning for each of the 20 CES-D items between more acculturated and less acculturated women was tested using ordinal logistic regression., Results: No items on the Depressed Affect, Somatic Activity, or Positive Affect subscales showed meaningful differential item functioning, but 1 item ("People were unfriendly") on the Interpersonal subscale had small results (R = 1.1%)., Discussion: The majority of CES-D items performed similarly for Spanish-speaking Hispanic women with high and low acculturation. Less acculturated women responded more positively to "People were unfriendly," despite having an equivalent level of depression, than did more acculturated women. Possibilities for improving this item are proposed.