We demonstrated the anti-inflammatory and anti-oxidative effects of Humulus lupulus (HL) extract on solar simulator-irradiated primary human keratinocytes (PHKs) by analyzing ERK and p38 MAPK phosphorylation and production of IL-6 and IL-8. The anti-inflammatory effect of topically applied HL was further tested in vivo on human skin. To this end, we developed an oil-in-water (O/W) and a water-in-oil (W/O) cream with a lipid content of 40%. The anti-inflammatory effect of 1% HL extract incorporated in these two vehicles was assessed in a randomized, prospective, placebo controlled, double-blind UVB erythema study with 40 healthy volunteers. Hydrocortisone acetate (HCA) in the corresponding vehicle served as positive control. Surprisingly, both HL and HCA were only effective in the O/W system but not in the W/O formulation. Release studies using vertical diffusion cells (Franz cells) revealed that HCA was released in much higher amounts from the O/W cream compared to the W/O formulation. In summary, we have shown that 1% HL extract exerts anti-inflammatory effects comparable to 1% HCA, but only when incorporated in our O/W cream. Our findings confirm the critical role of the vehicle in topical anti-inflammatory systems.