Understanding why the frequencies of some species’ vocalizations are far above or below those predicted by body size is key for explaining the remarkable diversity of mammalian vocal behavior. Horses, among the largest terrestrial mammals, provide a clear example of such deviation: their whinnies contain a very high fundamental frequency (>1,000 Hz) in addition to a second, lower one (∼200 Hz). While the lower fundamental frequency of whinnies is readily attributed to vocal fold (laryngeal) vibrations, the biomechanical processes underlying the production of the higher one remain unknown. Using a combination of in vivo and ex vivo data, including excised larynx experiments with helium, computed tomography (CT) scans, endoscopic examinations, and acoustic analysis of horses with recurrent laryngeal neuropathy, we provide evidence that the high fundamental frequency in horse whinnies is generated by an aerodynamic whistle mechanism within the larynx, rather than vocal fold tissue vibration. These separate laryngeal sources explain the simultaneous production of low and high fundamental frequencies in vocalizations (i.e., biphonation). Horse biphonation likely evolved to convey multiple independent messages concurrently, highlighting the role of anatomical and aerodynamic adaptations in enhancing vocal complexity across species.