In normal human skin, three peptidylarginine deiminases (PAD1-3) are expressed. To analyze PAD1 involvement during keratinocyte differentiation, this isotype was down-regulated by an RNA interference approach in a three-dimensional reconstructed human epidermis (RHE) model. During normal RHE production, from day 4 to day 10, the deimination rate increased by a factor of ~ 14 and PAD1 relative mRNA level by a factor of ~8. Like in interfollicular epidermis, deiminated proteins were mainly immuno-detected at the stratum corneum level. Down-regulation of PAD1 in this model, using two distinct shRNAs induced a reduction of keratohyalin granule area, a clear reduction of filaggrin monomer amount, a decrease of the number of corneocyte layers, and a quasi- full extinction of the protein deimination without major impact on keratinocyte proliferation. PAD1 deficiency altered the outside/in permeability of the epidermal model and induced the collapse of the trans-epidermal-electric resistance values without impact on the superficial pH or the trans-epidermal water-loss. At the protein and transcript levels, detection of cornified envelope constituents and autophagic markers was not affected but several tight junction actors were modulated and transglutaminases 1, 3 and 5 falled down. Furthermore, PAD1 deficiency also altered the shape of nuclei in the granular layer of RHEs, with deep invaginations of the nuclear membrane and a huge increase of perinuclear vesicles, as shown using transmission electron microscopy. Treatment of normal RHEs, from day 8 to day 10, with Cl-amidine, a pan PAD inhibitor, confirmed the involvement of PADs in nucleophagy. Therefore, PAD1 deficiency affects the epidermal barrier organization and function. The present work is also the first demonstration that PAD1 and deimination play a major role in the nucleophagy process associated with the late steps of keratinocyte differentiation.