Background: Hispanic women are disproportionately affected by gestational diabetes mellitus (GDM), yet few studies have assessed the impact of acculturation on health behaviors that may reduce GDM risk. Materials and Methods: We assessed relationships between acculturation and meeting American Diabetes Association guidelines for macronutrient intake and American College of Obstetricians and Gynecologists guidelines for physical activity (PA) using baseline data from Estudio Project Aiming to Reduce Type twO diabetes, a randomized trial conducted in Massachusetts (2013-2017) among 255 Hispanic pregnant women with hyperglycemia. Acculturation was assessed via the Psychological Acculturation Scale, duration of time and generation in the continental United States, and language preference; diet with 24-hours dietary recalls; and PA with the Pregnancy Physical Activity Questionnaire (PPAQ). Results: The majority of participants who reported low psychological acculturation (74.9%), preferred English (78.4%), were continental U.S. born (58.0%), and lived in the continental United States ≥5 years (91.4%). A total of 44.8%, 81.8%, 22.9%, and 4.6% of women met guidelines for carbohydrate, protein, fat, and fiber intakes, respectively; 31.9% met guidelines for PA. Women with higher acculturation were less likely to meet carbohydrate guidelines (English preference: adjusted risk ratios [aRR] 0.45, 95% confidence intervals [CI] 0.23-0.75; U.S. born: aRR 0.60, 95% CI 0.36-0.91; duration of time in United States: aRR 0.96, 95% CI 0.92-0.99). Women with higher acculturation were more likely to meet PA guidelines (U.S. born: aRR 1.95, 95% CI 1.11-3.44). Conclusions: In summary, higher acculturation was associated with lower likelihood of meeting dietary guidelines but greater likelihood of meeting PA guidelines during pregnancy. Interventions aimed at reducing GDM in Hispanics should be culturally informed and incorporate acculturation. Clinical Trial Registration: clinicaltrials.gov NCT01679210.