This pilot study explored physiological responses to playing and listening to the Native American flute. Autonomic, electroencephalographic (EEG), and heart rate variability (HRV) metrics were recorded while participants (N = 15) played flutes and listened to several styles of music. Flute playing was accompanied by an 84% increase in HRV (p < .001). EEG theta (4-8 Hz) activity increased while playing flutes (p = .007) and alpha (8-12 Hz) increased while playing lower-pitched flutes (p = .009). Increase in alpha from baseline to the flute playing conditions strongly correlated with experience playing Native American flutes (r = +.700). Wide-band beta (12-25 Hz) decreased from the silence conditions when listening to solo Native American flute music (p = .013). The findings of increased HRV, increasing slow-wave rhythms, and decreased beta support the hypothesis that Native American flutes, particularly those with lower pitches, may have a role in music therapy contexts. We conclude that the Native American flute may merit a more prominent role in music therapy and that a study of the effects of flute playing on clinical conditions, such as post-traumatic stress disorder (PTSD), asthma, chronic obstructive pulmonary disease (COPD), hypertension, anxiety, and major depressive disorder, is warranted.