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This study investigated the effectiveness of individual laryngeal muscles in regulating the mean glottal flow and glottal closure pattern during phonation in an in vivo canine larynx model. Phonation experiments were performed with parametric stimulation of the thyroarytenoid (TA), lateral cricoarytenoid (LCA), interarytenoid (IA), and the cricothyroid (CT) muscles. For each stimulation level, the subglottal pressure was gradually increased to produce phonation. The subglottal pressure, the volume flow, and the outside acoustic pressure were measured together with high-speed recording of vocal fold vibration from a superior view. The results show that the TA muscle played a dominant role in regulating both the membranous glottal width and the glottal closure pattern during phonation, indicating an important role of the TA muscle in regulating voice quality. The TA muscle activation was also the most effective in regulating the mean glottal flow, and thus an important laryngeal adjustment in airflow conservation, particularly at high subglottal pressures or loud voice production, although increasing TA activation decreased the vocal intensity. This study also presented a complete set of data on muscular control of the glottal width and voice production, which can be used in validation of computational models of vocal fold posturing and voice production.