A record number of Latino students are enrolling in higher education in the U.S., but as a group Latinos are the least likely to complete a bachelor’s degree. Cultural factors theoretically contribute to Latino students’ success, including orientation toward ethnic heritage and mainstream cultures (i.e., dual cultural adaptation), feeling comfortable navigating two cultural contexts (i.e., biculturalism), and the degree of fit between students’ cultural backgrounds and the cultural landscapes of educational institutions (i.e., cultural congruity). In a two-part study, these cultural factors were examined in relation to the hypothalamic-pituitary-adrenal (HPA) axis stress response (indexed by salivary cortisol), a physiological mechanism that may underlie how psychosocial stress influences academic achievement and health. First, Latino students’ cortisol responses to stress were estimated in their daily lives prior to college using ecological momentary assessment (N = 206; 64.6% female; Mage = 18.10). Results from three-level growth models indicated that cortisol levels were lower following greater perceived stress than usual for students endorsing greater Latino cultural values (e.g., familism), compared to students endorsing average or below-average levels of these values. Second, cortisol and subjective responses to a standard public speaking stress task were examined in a subsample of these same students in their first semester of college (N = 84; 63.1% female). In an experimental design, viewing a brief video prior to the stress task conveying the university’s commitment to cultural diversity and inclusion (compared to a generic campus tour) reduced cortisol reactivity and negative affect for students with greater Latino cultural values, and also reduced post-task cortisol levels for students with greater mainstream U.S. cultural values (e.g., competition). These findings join the growing science of culture and biology interplay, while also informing initiatives to support first-year Latino students and the universities that serve them.