We tested whether multi-walled carbon nanotubes (MWCNTs) induce oxidative stress and a pro-inflammatory response in human N-hTERT telomerase-immortalized keratinocytes, in human SZ95 SV-40 immortalized sebocytes and in in vitro reconstructed epidermises. MWCNTS were tested in various dispersion states, from raw and agglomerated particles to isolated entities obtained by sonication in the presence of dispersive agents (hydroxypropylcellulose and Pluronic F108). It was observed that: (a) Contrary to individualized MWCNTs, agglomerated particles prepared by suspension into pure water increased the intracellular levels of reactive oxygen species as well as the expression and secretion of interleukin-8 in N-hTERT cells; (b) the inflammatory signature of MWCNTs in N-hTERT cells, drawn by transcriptomic analysis with low-density microfluidic cards, included various other cytokines such as interleukin-6 or C-C motif ligand 3; (c) the pro-inflammatory effects of MWCNTs, as assessed by interleukin-8 transcript level and protein release, were not observed in SZ95 cells; and (d) the secretion of interleukins-1α and -8 from in vitro reconstructed epidermal tissues, used as specific markers for skin irritation and sensitization, was unaffected in presence of MWCNTs, confirming that the cornified layer is an efficient barrier against MWCNTs.