Whistling has become more popular in recent years. “Whistling competitions” are being held in Japan since 2005. However, it can be difficult to teach some people how to whistle. One of the reasons is that theoretical information, including clear descriptions about how to whistle is scant. A better understanding of the principle would be beneficial to both the trainer and the trainee. In this study, the glottis opening area during whistling was measured via endoscopic imaging data. The amplitude characteristics of the vocal tract were determined by measuring the glottis opening area during whistling and calculating the transfer function of the vocal tract in conjunction with that area. By considering the glottis opening area, we could logically explain the fact that, in reality, some notes corresponding to theoretically calculated resonance frequencies did not occur, and we could obtain a calculation result that was close to the physical model. Furthermore, because the calculation result close to the physical model was only associated with certain glottis opening areas, it is evident that not only the shape of the vocal tract and the lip opening area, but also the glottis opening area is an important component of sound production during human whistling.