PURPOSE: We evaluated the associations between accelerometer-estimated physical activity (PA) intensity and heart rate variability (HRV) and examined mediation of these associations by glycemic control indices and other cardiovascular disease risk factors. METHODS: Data were from 1668 participants (X[Combining Overline]age = 45.9 ± 3.5 yr, 58.0% female, 39.9% black) who participated in year 20 (2005-2006) of the Coronary Artery Risk Development in Young Adults Fitness Study. The ActiGraph 7164 estimated participants' mean minutes per day of vigorous-intensity PA (VPA), moderate-intensity PA (MPA), and light-intensity PA (LPA) over 7 d. Three sequential 10-s 12-lead ECG strips were used to derive standard deviation of all normal RR intervals (SDNN) and root mean square of all successive RR intervals (rMSSD) HRV. Mediators representing glycemic control indices included fasting glucose, fasting insulin, and 2-h oral glucose tolerance, with other mediators being traditional cardiovascular disease risk factors. Multiple linear regression assessed independent associations of PA intensity with HRV per 1-SD. Mediation analyses computed the proportion of the PA-HRV association attributable to physiological mediators. RESULTS: Participants averaged 2.7 ± 6.2 min·d, 33.0 ± 22.0 min·d, and 360.2 ± 83.8 min·d of VPA, MPA, and LPA, respectively, with mean values for SDNN (32.6 ± 22.4 ms) and rMSSD (34.0 ± 24.8 ms) similar. After adjustment for demographic and lifestyle behaviors, VPA was associated with both HRV metrics (SDNN: std beta = 0.06 [0.03, 0.10]; rMSSD: std beta = 0.08 [0.05, 0.12]) and LPA with rMSSD only (std beta = 0.05 [0.01, 0.08]). Fasting insulin and glucose mediated 11.6% to 20.7% of the association of VPA and LPA with HRV, with triglycerides also potentially mediating these associations (range, 9.6%-13.4%). CONCLUSIONS: Accelerometer-estimated VPA was associated with higher (i.e., improved) HRV. Light-intensity PA also demonstrated a positive association. Mediation analyses suggested these associations may be most attributable to glucose-insulin dynamics.