This preregistered, randomized field experiment tested the effectiveness of a brief deep breathing intervention on children's concurrent physiological arousal in naturalistic settings (N  = 342; Mage = 7.48 years; 46% female; 53% Asian, 26% White; 21% other race/ethnicity). The treatment consisted of an animated video that introduced deep breathing as a self-regulation strategy and scaffolded the child in taking a few slow-paced breaths, while the control group watched an informational video featuring similar animated images. Respiratory sinus arrhythmia (RSA) and heart rate (HR) were measured while children were sitting still (baseline) and subsequently while watching 1-min videos. Relative to baseline arousal, RSA increased and HR decreased only in response to the deep-breathing treatment video. Effects were larger in the second 30-s epoch of the video, which included most of the deep breathing practice. RSA fully mediated the intervention's effects on HR. By analyzing all children exposed to intervention video regardless of their engagement in the deep breathing practice (intention-to-treat design) and by using easily scalable treatment videos, the study identifies an effective and pragmatic approach to reducing children's physiological arousal in everyday, group settings. Implications for advancing applied developmental psychophysiological research are discussed.