Transdermal delivery of nanoparticles is by most part limited by the stratum corneum, a thin barrier located within the epidermis that serves to limit molecules from entering the skin. Although there have been reports on the penetration of gold nanoparticles through intact skin in vitro, little is known regarding their penetration efficiency in vivo with regards to size and shape. The present work aims to use an in vivo rat model to elucidate how PEG-OAm conjugated ultra-small gold nanoparticles such as nanospheres and nanorods interact with and penetrate the skin depending on these two factors. Confocal microscopy (CLSM) and transmission electron microscopy (TEM) methods were used to study the localization of modified gold nanoparticles into rat skin after topical application. CLSM micrographs revealed that the permeation property of ultra-small gold nanoparticles was independent of size but was correlated to shape. The deposition of gold nanorods in the epidermis was 1.9 times that of gold nanospheres and the accumulation of rods in the subcutaneous adipose tissue, a marker for skin penetration was 1.7-times that of spheres. Moreover, TEM micrographs of the subcutaneous adipose tissue showed the presence of gold nanoparticles in the subcutaneous adipose tissue. The study herein provides insights to the in vivo permeation of ultra-small gold nanoparticles and demonstrates their potential for topical delivery.