We studied the use of acoustics for coalescence of water droplets and dehydration of crude oil emulsion. Experimental studies were conducted using acoustic standing waves in a resonant cavity to trap water droplets and enhance oil separation. The focus was on the effect of ultrasound irradiation on crude oil emulsion properties, such as viscosity, water drop radius, shear strength of oil-water interfacial films, and flocculation size of asphaltene. These properties are important to the coalescence of water drops in water-oil (W/O) emulsion in the process of oil separation with ultrasound. Ultrasound irradiation is able to decrease the emulsion stability, which provides a new insight into the acoustics-aided demulsification mechanism. It can be considered as a supplement of traditional acoustics-aided demulsification mechanism (ultrasound-induced motion of water droplets). Furthermore, the effects of ultrasonic parameters such as the type of ultrasonic field, irradiation time, frequency, and acoustic intensity on dewatering the W/O emulsion are discussed. These results provide guidance for setting the optimum conditions for the separation of W/O emulsion with ultrasound. Under the optimum conditions, water content in crude oil emulsion can be decreased from 40% to 3.8%, which satisfies the requirement of dehydration for refinery.