While ultrasound-guided injections are safer and more effective than the typical (blind) injection methods, there has been no report of ultrasound-guided injection techniques being applied to patients for aesthetic treatment or to patients with an asymmetrical smile. The purpose of this study was to determine the locations of facial expression muscles using ultrasound and to use this information to guide BoNT-A injections into a patient with an asymmetrical smile. The safety and effectiveness of this technique were also evaluated. A real-time two-dimensional B-mode ultrasound system (E-CUBE 15 EX, ALPINION, Seoul, Korea) with a 30-mm-wide linear array transducer (8.0-17.0 MHz; IO8-17 high frequency hockey stick, Seoul, Korea) was used to obtain images of the depressor labii inferioris (DLI), risorius, depressor anguli oris (DAO), and zygomaticus major (ZMj). The patient received an ultrasound-guided BoNT-A injection (Meditoxin®, Medytox, Seoul, Korea) in each target muscle using a 1-mL syringe with a 30-gauge 1-inch-long needle. The patient’s smile was symmetrical 1 month after the US-guided BoNT-A into the DLI, risorius, and ZMj. The symmetry was achieved by decreasing the exposure of the lower teeth and by elevating both mouth corners. The ultrasound-guided injection technique may contribute to the determination of the optimal injection method and dosage based on a precise knowledge of morphology, location, layer of target muscles, and thickness of subcutaneous fat tissues.