The aim of this work was the preparation, characterization, and comparison of novel pH-sensitive nonphospholipid vesicles (niosomes) from two nonionic surfactants, with different hydrophilic-lipophilic balance values (Tween20-TW20 = 16.7 and Span60-SP60 = 4.7). Surfactants were mixed with cholesterol (CHOL) and its derivative, cholesteryl hemisuccinate (CHEMS), as a pH-sensitive molecule. Vesicles were characterized by dynamic light scattering, in order to evaluate their dimensions and vesicle stability, by zeta-potential measurements and by means of electronic microscopy after freeze-fracture. Ibuprofen (IBU) was used as the model drug, and high-performance liquid chromatography analyses were performed to evaluate drug-entrapment efficiency and release in a neutral, acidic environment. The influence of the vesicle composition on skin accumulation and transdermal permeation of IBU across excised hairless rat skin was investigated by using vertical Gummer diffusion cells. When niosomes with SP60 and CHEMS were prepared, there was a statistically significant increase of skin permeation of IBU, while TW20 niosomes did not show statistically significant differences in P(app) values without the influence of the vesicle size and charge.