Background/Aims: The skin provides protection against chemical, physical, and biological stressors, yet the skin morphology changes over the course of life. These changes might affect the skin barrier function and facilitate the onset of age-related diseases. Since orally applied lactic acid bacteria ameliorate signs of aged and atopic skin, we investigated the effects of a topically applied Lactococcus lactis emulsion. Methods: In a blinded, randomized, vehicle-controlled trial, we studied topical Lactococcus effects both in vitro and in 20 healthy female volunteers. Commercially available reconstructed human epidermis (RHE) was treated for 4 days (once daily) and volar forearms were treated for 30 days (twice daily). Results: Lactococcus formulations improve the skin barrier in RHE as shown by increased filaggrin and human β-defensin-2 expression as well as by the 23% declined mean apparent permeability coefficients for caffeine. A reduction of 18% in transepidermal water loss confirms this effect in humans. Moreover, Lactococcus emulsion optimized skin hydration and surface pH. Skin irritation was not detected. Conclusions: Lactococcus emulsion improved the skin barrier function with good biocompatibility. Moreover, our study exemplifies the translational predictive capacity of testing on RHE with respect to Lactococcus emulsion.