Our previous studies demonstrated the ultrasound-induced skin optical clearing enhancement with topical application of optical clearing agents on in vitro porcine skin and in vivo human skin. The objective of this study was to investigate the possible mechanisms of the enhanced skin optical clearing by ultrasound medications. Optical clearing effects of ex vivo guinea pig abdomen skin topically applied with 60% glycerol or the combination of 60% glycerol and ultrasound were studied by optical coherence tomography (OCT). Microstructure of skin surface was examined by scanning electron microscopy (SEM). Ultrasound with a frequency of 1MHz and a power of 0.75W over a 3-cm probe was simultaneously applied with glycerol solution for 15min. The combination of 60% glycerol and ultrasound results in a 19% increase in OCT 1/e light penetration depth after 30min, which is much better than 60% glycerol alone. SEM images demonstrated that changes in skin microstructure due to the tight order of the lipid bilayers in the stratum corneum disrupted and the separation of keratinocytes by the application of ultrasound contribute to the ultrasound-enhanced intact skin optical clearing effects.