Background. Less than 50% of Latinas meet physical activity (PA) recommendations. Acculturation is a complex cultural phenomenon that may influence health behaviors, but associations between acculturation and Latinas’ activity and sedentary levels are unclear. Aim. To examine associations of acculturation with Latinas’ domain-specific and total PA as well as sedentary time. Method. We analyzed baseline data collected between 2011 and 2013 among 410 Latinas (18-65 years) from a PA promotion intervention in San Diego, CA (Fe en Acción/Faith in Action). Participants wore an accelerometer to assess moderate-to-vigorous PA (MVPA) and sedentary time and completed a survey assessing domain-specific PA, sociodemographics, and acculturation as measured by length of residence in the United States and the Bidimensional Acculturation Scale (BAS) for Hispanics. Higher acculturation was defined as longer residence in the United States or being either assimilated or bicultural as per scores on the Hispanic and Anglo domains of the BAS. Results. Based on weekly averages from the accelerometer, Latinas spent 103 minutes in MVPA and 76% of total activity in sedentary time. Only 32% met MVPA recommendations via self-reported leisure-time and transportation PA. Longer residence in the United States was inversely associated with reporting any transportation or occupational PA and meeting MVPA recommendations. Assimilated/bicultural Latinas had significantly less accelerometer-based total MVPA and higher sedentary time than their lower acculturated counterparts. Conclusions. Overall, higher acculturation, based on either measure, was related to less activity. Our findings suggest interventions tailored to the acculturation levels of Latinas are needed to help reduce disparities in Latinas’ PA and sedentary behaviors.