Airway epithelial cilia protect the mammalian respiratory system from harmful inhaled materials by providing the force necessary for effective mucociliary clearance. Ciliary beating is asymmetric, composed of clearly distinguished effective and recovery strokes. Neither the importance of nor the essential components responsible for the beating asymmetry has been directly elucidated. We report here that the beating asymmetry is crucial for ciliary function and requires tubulin glutamylation, a unique posttranslational modification that is highly abundant in cilia. WT murine tracheal cilia have an axoneme-intrinsic structural curvature that points in the direction of effective strokes. The axonemal curvature was lost in tracheal cilia from mice with knockout of a tubulin glutamylation-performing enzyme, tubulin tyrosine ligase-like protein 1. Along with the loss of axonemal curvature, the axonemes and tracheal epithelial cilia from these knockout (KO) mice lost beating asymmetry. The loss of beating asymmetry resulted in a reduction of cilia-generated fluid flow in trachea from the KO mice. The KO mice displayed a significant accumulation of mucus in the nasal cavity, and also emitted frequent coughing- or sneezing-like noises. Thus, the beating asymmetry is important for airway ciliary function. Our findings provide evidence that tubulin glutamylation is essential for ciliary function through the regulation of beating asymmetry, and provides insight into the molecular basis underlying the beating asymmetry.