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ObjectiveMindfulness interventions, which train practitioners to monitor their present-moment experience with a lens of acceptance, are known to buffer stress reactivity. Little is known about the active mechanisms driving these effects. We theorize that acceptance is a critical emotion regulation mechanism underlying mindfulness stress reduction effects. Method In this three-arm parallel trial, mindfulness components were dismantled into three structurally equivalent 15-lesson smartphone-based interventions: (1) training in both monitoring and acceptance (Monitor + Accept), (2) training in monitoring only (Monitor Only), or (3) active control training (Coping control). 153 stressed adults (mean age = 32 years; 67% female; 53% white, 21.5% black, 21.5% Asian, 4% other race) were randomly assigned to complete one of three interventions. After the intervention, cortisol, blood pressure, and subjective stress reactivity were assessed using a modified Trier Social Stress Test. Results As predicted, Monitor + Accept training reduced cortisol and systolic blood pressure reactivity compared to Monitor Only and control trainings. Participants in all three conditions reported moderate levels of subjective stress. Conclusions This study provides the first experimental evidence that brief smartphone mindfulness training can impact stress biology, and that acceptance training drives these effects. We discuss implications for basic and applied research in contemplative science, emotion regulation, stress and coping, health, and clinical interventions.