Aim of this work was to prepare and characterize a hydroxyethyl cellulose hydrogel loaded with ascosomes, nanovesicles based on phosphatidylcholine plus ascorbyl octanoate (ASC8) or ascorbyl decanoate (ASC1), and khellin (2 mg/mL), for topical use. ASC10 vesicles were selected for the hydrogel formulation because of the best biopharmaceutical characteristics, namely size of 115 nm, PDI of 0.26, ζ-potential of -40.1 meV, EE% of 90.2%. After 24 h the in vitro release of khellin was more than 80%, while the ex-vivo skin permeation of khellin after application of the vesicles was 42% of the dose. The hydrogel formulations had a pH value of 5, viscosity properties were different according to the different temperatures and in addition, they presented characteristics of non-Newtonian fluids with a pseudoplastic shear thinning behaviour according to the Herschel-Bulkley equation. These hydrogels combine the advantages of a suitable viscosity for dermal use (hydrogel matrix) and an increased transdermal absorption (ascosome components). The best permeability of the ASC10 ascosomes, led to select the formulation for skin irritation and corrosion tests in rats. Liver and dermal histological and pathological analyses demonstrated that hydroxyethyl cellulose hydrogels based on khellin loaded in the ASC10 ascosomes have no toxic effects.