Mind–body interventions, such as mindfulness-based stress reduction (MBSR), may improve well-being by increasing awareness and regulation of physiological and cognitive states. However, it is unclear how practice may alter long-term, baseline physiological processes, and whether these changes reflect improved well-being. Using respiration rate (RR), which can be sensitive to effects of meditation, and 3 aspects of self-reported well-being (psychological well-being [PWB], distress, and medical symptoms), we tested pre-registered hypotheses that: (1) Lower baseline RR (in a resting, non-meditative state) would be a physiological marker associated with well-being, (2) MBSR would decrease RR, and (3) Training-related decreases in RR would be associated with improved well-being. We recruited 245 adults (age range = 18–65, M = 42.4): experienced meditators (n = 42), and meditation-naïve participants randomized to MBSR (n = 72), active control (n = 41), or waitlist control (n = 66). Data were collected at pre-randomization, post-intervention (or waiting), and long-term follow-up. Lower baseline RR was associated with lower psychological distress among long-term meditators (p* = 0.03, b = 0.02, 95% CI [0.01, 0.03]), though not in non-meditators prior to training. MBSR decreased RR compared to waitlist (p = 0.02, Cohen’s d = − 0.41, 95% CI [− 0.78, − 0.06]), but not the active control. Decreased RR related to decreased medical symptoms across all participants (p* = 0.02, b = 0.57, 95% CI [0.15, 0.98]). Post-training, lower RR was associated with higher PWB across training groups compared to waitlist (p* = 0.01, b = 0.06, 95% CI [0.02, 0.10]), though there were no significant differences in change in PWB between groups. This physiological marker may indicate higher physical and/or psychological well-being in those who engage in wellness practices.