Prior research has demonstrated that the daily experience of negative affect is associated with increased levels of proinflammatory activity as evidenced by higher interleukin-6 among Americans but not among Japanese. This cultural difference may be driven by culturally divergent beliefs about negative affect as a source of threat to self-image versus as natural and integral to life. Here, we examined whether culture may moderate the relationship between negative affect and biological stress responses, with a focus on the hypothalamic-pituitary-adrenal (HPA) axis activity. By using culturally matched surveys of Americans (N = 761) and Japanese (N = 328), we found that negative affect was associated with a flattening of the diurnal cortisol slope among Americans after controlling for demographic variables, personality traits, sleep patterns, and health behaviors. In contrast, the association between negative affect and the HPA axis activity was negligible among Japanese. Moreover, we assessed biological health risk with biomarkers of both inflammation (interleukin-6 and C-reactive protein levels) and cardiovascular function (higher systolic blood pressure and total-to-HDL cholesterol ratio) and found that the relationship between negative affect and increased biological health risk, which was observed only among Americans, was mediated by the flattening of the diurnal cortisol rhythm. These findings suggest that cultural differences in how emotions are construed may make the experience of negative affect more or less stressful and differentially consequential for health. (PsycInfo Database Record (c) 2020 APA, all rights reserved).