The Acceptance and Action Questionnaire-II (AAQ-II) is a widely used measure of experiential avoidance and psychological inflexibility, two key constructs of acceptance and commitment therapy (ACT). The main aim of this study was to analyze the psychometric properties and factor structure of the Spanish version of the AAQ-II in Colombia. A secondary aim was to analyze measurement invariance of the AAQ-II across nonclinical and clinical samples considering that previous research has yielded mixed results. The AAQ-II was administered to a total of 1759 participants, including a sample of undergraduates, one of general population, and a clinical sample. Data were very similar to the ones obtained in the original AAQ-II version. The internal consistency across the different samples was good (Cronbach’s alpha between 0.88 and 0.91). The one-factor model found in the original scale showed a good fit to the data. Measurement invariance was also found across sample (clinical and nonclinical) and gender. The mean score of the clinical sample on the AAQ-II was significantly higher than the scores of the nonclinical samples. The AAQ-II was sensitive to the effects of a one-session ACT intervention, and AAQ-II scores were significantly related to emotional symptoms, mindfulness, satisfaction with life, and dysfunctional attitudes. The Spanish version of the AAQ-II shows good psychometric properties in Colombia.